WO2004061107A1 - Cell growth inhibitor using sphingosine kinase 2, method of constructing fused protein having its nuclear localization signal, method of screening drug candidate, and screening kit - Google Patents

Abstract

It is intended to provide medically and industrially useful substances and methods based on the analysis of the function of sphingosine kinase 2 (SPHK2). As the results of the analysis on the function of sphingosine kinase 2 (SPHK2), it is found out that SPHK2 occurs mainly in the nucleus of a cell and has a nuclear localization signal (NLS) in its amino-terminal side, DNA synthesis in cells is inhibited by expressing SPHK2, SPHK2 should be present in the nucleus to exert its effect of inhibiting the DNA synthesis, etc. Therefore, SPHK2 is usable as a cell growth inhibitor and its NLS sequence is applicable to the construction of a novel fused protein or the like, which makes them medically and industrially useful.

Description

 Description Cell growth inhibitor using sphingosine kinase 2, a method for producing a fusion protein having a nuclear translocation signal thereof, a method for screening a drug candidate substance, and a screening kit The present invention relates to a cell growth inhibitor using sphingosine kinase 2, a method for producing a fusion protein having a nuclear localization signal, and a method for screening a drug candidate.

BACKGROUND ART This application is a US Provisional Application Serial No. (U.S. Provisional Application Serial No.) 60 / 484,348 (filing date: July 1, 2003, title of invention: Sphingosine kinase 2 is a Nuclear protein and mhioits DNA synthesis, and claims the benefit of the US provisional patent application. Sphingosine 1-posphate; S

PP) acts as a lipid activator or lipid mediator inside and outside the cell and regulates various functions of the cell. It has been reported at least to be involved in a wide variety of physiological functions such as cell proliferation, angiogenesis, suppression of apoptosis, cell differentiation, cell motility, and cytoskeletal rearrangement. The above-mentioned sphingosine monophosphate is EDG (Endothelial Differentiation) Gene) It has recently been shown to be a ligand for the receptor family (also called the “SPP receptor”), and sphingosine 1-phosphate has been shown to act as an extracellular ligand. To date, at least five receptors for sphingosine 1-phosphate (i.e., EDG-1 / SPPI, EDG-5 / SPP2, EDG-3 / SPP3, EDG-6 / SPP4, EDG-8 / SPP5) has been identified.

 In addition, sphingosine 1-phosphate has a role as an intracellular second messenger and is known to play an important role in the release of calcium from intracellular storage areas and suppression of apoptosis. ing. On the other hand, sphingosine kinase (hereinafter also referred to as “SPHK”) is an enzyme that phosphorylates sphingosine and produces the above-mentioned sphingosine 1-phosphoric acid, and reduces the amount of sphingosine 1-phosphoric acid in cells. Adjust. Two isoforms of sphingosine kinase 1 and sphingosine kinase 2 (hereinafter also referred to as “SPHK1” and “SPHK2”, respectively) are known as SPHKs derived from mammals (for example, Kohama et al. Biol. Chem. (1998) 273, 23722-23728; Liu et al. J. Biol. Chem. (2000) 275, 19513-19520). Among them, the amino acid sequence of SPHK2 and its base sequence are described in the following databases (I) and (II).

(I) DDBJ: session number AF245447 (Homo sapiens)

)

(II) DDBJ: Accession No. AF245448 (Mus muscu lus) There are differences between SPHK1 and SPHK2 in their biochemical properties. For example, in vitro, the activity of SPHK1 is strongly activated by the detergent Triton X-100, but suppressed by salts. In contrast, the activity of SPHK2 shows the opposite reaction. When SPHK1 is expressed in 293 cells, the enzyme activity is dramatically increased, but when SPHK2 is expressed, it is not so increased.

 As a result of functional analysis, the above SPHK1 showed that, when overexpressed, it could (1) promote the transition of cells from G1 to S phase and induce cell proliferation, and (2) suppress the apoptotic response of cells. Are known. It has also been reported that overexpression of SPHK1 causes NOD / SCID mice to form tumors. Furthermore, multiple molecules such as TRAF2, RPK118, and ΑΚΑ-related proteins have already been identified as SPHK1-binding molecules.

 On the other hand, little is known about the function of SPHK2. As described above, sphingosine 1-phosphate, a substrate of SPHK2, is involved in a wide variety of physiological functions such as cell proliferation, angiogenesis, and suppression of apoptosis, and as a result, various diseases related to these physiological functions (eg, , Arteriosclerosis, essential hypertension, etc.). Therefore, elucidating the function of the phosphorylation enzyme SPHK2 will lead to the analysis of the pathology of such diseases and the development of diagnostic and therapeutic methods, and will have important medical and industrial contributions. Be expected.

The present invention has been made in view of the above problems, and an object of the present invention is to provide a method and a substance which are medically and industrially useful by performing functional analysis of SP # 2 and analyzing the function. Disclosure of the invention

In view of the above problems, the present inventor has conducted detailed functional analysis of SPHK2. As a result, SPHK2 is mainly present in the nucleus in cells, and a nuclear translocation signal (NLS) Expression of SPHK2 suppresses cell DNA synthesis, and that SPHK2 must be present in the nucleus in order to exert its DNA synthesis inhibitory effect. The present invention has been completed.

 That is, the present invention includes the following inventions A) to J) as medically or industrially useful methods and materials.

 A) A cell growth inhibitor containing a gene encoding a mammalian sphingosine kinase 2 protein or its protein.

B) The cell growth inhibitor of the above A), wherein the cell growth inhibitor comprises sphingosine kinase 2 derived from human or mouse.

 C) An oligonucleotide encoding a nuclear transport signal located on the amino-terminal side of mammalian sphingosine kinase 2.

 D) A method for producing a fusion protein by fusing the nuclear transport signal of sphingosine kinase 2 with another protein using the oligonucleotide described in C) above.

 E) A fusion protein produced by the method described in D) above.

 F) The fusion protein according to E) above, wherein the nuclear transfer signal of sphingosine kinase 2 and sphingosine kinase 1 are fused.

G) The fusion protein described in F) above, or the fusion protein A cell growth inhibitor containing an encoding gene.

 H) A method for screening a substance that regulates the cell growth inhibitory action of sphingosine kinase 2.

I) The screening method according to H) above, wherein a full-length or partial protein of sphingosine 5 kinase 2 or a modified protein thereof, or a gene encoding these proteins is used. Ning method.

 J) A drug containing a substance obtained by the screening method described in H) or I) above.

 • 0 K) Screening kit for implementing the screening method described in H) above.

 L) The screening kit described in the above) is characterized in that it contains the full-length or partial protein of sphingosine kinase 2, a modified form of the protein, or a gene encoding these proteins. Screening kit.

 Further objects, features, and advantages of the present invention will be made clear by the description below. Also, the advantages of the present invention will become apparent in the following description with reference to the accompanying drawings. 0 Brief description of the drawings

FIG. 1 shows the results of examining the intracellular distribution of SPHK2 and SPHK1. Fig. 2 (a) is a diagram schematically showing the structures of mSPHKl, mSPHK2, mSPHK2AM, and mSPHK2A N. Fig. 2 (b) is a result of examining the intracellular distribution of mSPHK2, mSP m2ΔΗ, and mSPHK2A N. FIG.

 Fig. 3 (a) shows the comparison between the NLS sequence of SPHK2 and the NLS sequences of other proteins.Fig. 3 (b) shows the intracellular distribution of SPHK2R93E / R94E and NLS-SPH K1. It is a figure showing a result.

 Figure 4 (a) is a graph showing the results of examining whether SPHK2 expression suppresses intracellular DNA synthesis.Figure 4 (b) shows that SPHK2 expression is induced according to Dox concentration. FIG. 9 is a diagram showing the result of an immunoplot analysis that has been confirmed.

 FIG. 5 is a graph showing the results obtained by expressing SPHK2, SPHK1, or a variant thereof in a cell, and examining how DNA synthesis in the cell changes.

 Figure 6 (a) shows the results of examining the presence or absence of intracellular expression of SPHK2 in each cell cultured in the presence or absence of Dox, and Figure 6 () shows the expression of SPHK2. FIG. 9 is a view showing the result of examining whether or not induces apoptosis.

 FIG. 7 (a) and FIG. 7 (b) show the results of examining how SPHK2 expression affects the cell cycle.

 FIG. 8 shows the results of examining the intracellular distribution of SPHK2, SPHK1, or the mutant.

FIG. 9 is a table showing the results of examining how the localization of SPHK2 changes depending on the cell type and cell density. Fig. 10 (a) shows the results of examining the activity of endogenous SPHK2, and Fig. 10 (b) shows the results of detection of the location of endogenous SPHK2 by immunlot. FIG. 10 (c) is a view showing the result of examining the intracellular distribution of endogenous SPHK2.

 Fig. 11 (a) is a graph showing the results of examining the activities of various SPHK proteins. Fig. 11 (b) shows the SPP in the cytoplasmic fraction (C) and the nuclear fraction (N). It is a graph which shows the result of having investigated accumulation.

 FIG. 12 shows the results of examining the intracellular distribution of the HA-SPHK2 protein using different epitope-specific antibodies.

BEST MODE FOR CARRYING OUT THE INVENTION

 Hereinafter, specific embodiments of the present invention will be described.

 (1) Cell growth inhibitor

 As described above, the cell growth inhibitor of the present invention is a cell growth inhibitor containing a mammalian sphingosine kinase 2 (SPHK2) protein or a gene (nucleic acid) encoding the protein.

 Here, "sphingosine kinase 2 (SPHK2)" means a protein mainly derived from human or mouse. SPHK2 derived from human is composed of 6 18 amino acids, and its amino acid sequence and nucleotide sequence are described in, for example, accession number AF245447. SPHK2 derived from mouse is composed of 6 17 amino acids, and its amino acid sequence and base sequence are described in, for example, accession number AF245448.

As shown in the examples below, expression of SPHK2 results in cellular DNA synthesis. Most of the cells transfected with SPHK2 remained in the G1 / S phase 9 hours after the G1 / S phase in the cell cycle (Fig. 4 (a), Fig. 4 (b) , Figure 5, Figure 7 (a) and Figure 7 (b)). Therefore, the SP HK2 protein and the gene encoding the protein can be used as a cell growth inhibitor. The SPHK2 protein is not limited to that derived from human or mouse, but may be any derived from mammals. Moreover, even if it is SPHK2 derived from human or mouse, it is not limited to the amino acid sequence described in the above accession number, but may be a part of the sequence. That is, SPHK2 derived from human or mouse includes not only (a) the protein consisting of the amino acid sequence described in the above accession number, but also (b) the amino acid sequence described in the above accession number. And a protein comprising an amino acid sequence in which one or several amino acids have been replaced, deleted, inserted, and / or added, and which has a DNA synthesis inhibitory action. The above-mentioned "one or several amino acids are substituted, deleted, inserted, and Z or added" means that the substitution, deletion, insertion, or substitution is performed by a known mutant protein production method such as site-directed mutagenesis. And Z or an addable number (preferably 10 or less, more preferably 7 or less, and more preferably 5 or less) of amino acids are substituted, deleted, inserted, and Z or added Means that As described above, the protein of (b) is a mutant protein of the protein of (a), and the “mutation” referred to here is mainly a mutation artificially introduced by a known mutant protein production method. However, it may be a protein obtained by isolating and purifying a similar naturally occurring mutant protein.

SPHK2 protein also contains additional polypeptides. You may use it. Such polypeptides may be added, for example, when the SPHK2 protein is epitopically labeled with His, Myc, Flag, or the like. Further, the SPHK2 protein may be modified by sugar chain binding or phosphorylation. The cell growth inhibitor of the present invention may contain a gene encoding SPHK2 protein, and the term "gene" as used herein includes at least an open library in the nucleotide sequence described in the above accession number. It includes a gene having one ding frame region and a modified gene obtained by modifying a part of its nucleotide sequence.

 The term "gene" includes DNA and RNA. The DNA includes, for example, cDNA and genomic DNA obtained by cloning or chemical synthesis techniques or a combination thereof. The DNA / RNA may be double-stranded or single-stranded, and the single-stranded strand may be a coding strand serving as a sense strand or an anti-coding strand serving as an antisense strand. Further, the “gene” may include a sequence such as an untranslated region (UTR) or a vector sequence (including an expression vector sequence) in addition to a coding sequence encoding a protein.

The cell growth inhibitor of the present invention may be any as long as it contains the above-described SPHK2 protein or the SPHK2 gene encoding the protein. In addition, an appropriate solution (including a buffer), an enzyme, a vector, Or, it may contain other chemical substances such as lipids, proteins, and low molecular weight compounds. The cell growth inhibitor of the present invention can be used in various cell culture techniques, in regenerative medicine represented by the preparation of tissues such as skin tissue, and in the preparation of organs and organs, and in real- It can be used for experimental systems. In addition, it can be applied to therapeutic drugs (especially for cell proliferative diseases).

 (2) Method for Producing Fusion Protein Having SPHK2 Nuclear Translocation Signal As shown in the Examples below, the present inventors have found that SPHK2 is mainly present in the nucleus in cells, By having a translocation signal (NLS) and expressing the NLS sequence fused to SPHK1 protein, SPHK1 protein that originally does not translocate to the nucleus can be translocated to the nucleus. The protein exhibited a DNA synthesis inhibitory effect that was not inherent in wild-type SPHK 1.

 Therefore, by fusing the nuclear translocation signal (NLS) of SPHK2 with other proteins, proteins that do not naturally translocate to the nucleus can be translocated to the nucleus, and proteins that do not naturally translocate to the nucleus can be translocated to the nucleus. Thus, it is possible to exert functions and actions that are not inherent in the wild type of the protein.

 Although the method for producing the fusion protein of the present invention having the nuclear localization signal (NLS) of SPHK2 is not particularly limited, the following briefly describes the production method used in Examples described later.

In the experiment, a fusion protein was prepared by fusing the NLS sequence of mouse-derived SPHK2 with the mouse-derived SPHK1 protein. The NLS sequence of SP HK2 derived from mouse was identified by the present inventor as the 87th to 95th amino acid sequence “RGRRGGRRR (SEQ ID NO: 1)” present on the amino terminal side. From comparison with other NLS sequences, it was concluded that the amino acid sequence “RRGGRR” of the 89th to 94th positions was particularly important in function. In preparing the fusion protein, an oligonucleotide (the following sense primer) encoding the above NLS sequence was used. First, the SPHK1 gene was cloned from the mouse brain cDNA library. Next, the plasmid into which the SPHK1 gene had been inserted was used as a type II gene for amplification by PCR. At this time, an oligonucleotide which encodes the above NLS sequence is used as a sense primer, and specifically, an oligonucleotide consisting of the following nucleotide sequence encoding the amino acid sequence of the 87th to 98th amino acids of SPHK2 derived from mouse. Nucleotides were used.

5 '-GCA AGA TCT CGT GGC CGT CGA GGG GGC CGG CGC AGA GCT ACG CGG A G GAA CCA GAA TGC- 3' (Distribution IJ number 2

) Of the above oligonucleotides, the 10th to 45th sequences correspond to the regions encoding the 87th to 98th amino acid sequences of SPHK2. The sequence at positions 46 to 60 corresponds to a region encoding the amino acid sequence at positions 1 to 5 of SPHK1. The amplified fragment obtained by the PCR method was inserted into pTB-701 beta to prepare an expression vector. This expression vector is transferred to a host cell (NIH in the examples).

3T3 cells) and expressed the fusion protein.

When a fusion protein having the NLS sequence of human-derived SPHK2 is prepared by the same method as described above, the NLS sequence is composed of the amino acid sequence at position 86 to 94 of human-derived SPHK2 `` RGRRGARIIR (SEQ ID NO: 3) ", and especially since the RRGARRJ of the 88th to 93rd amino acid sequence is considered to be functionally important, the oligonucleotide encoding this NLS sequence is used. It is good to use. Of course, the fusion protein of the present invention may be prepared by a method other than the above. 'Furthermore, since the fusion protein obtained by fusing the NLS system of SPHK2 with the SPHK1 protein exhibited a DNA synthesis inhibitory effect similarly to the SPHK2 protein, the fusion protein or the gene encoding the fusion protein was Like the cell growth inhibitor of the above (1), it can be used as a cell growth inhibitor.

(3) Screening method for drug candidate substances

 As described above, since the SPHK2 protein has a DNA synthesis inhibitory action (in other words, a cell growth inhibitory action), the SPHK2 protein and its gene can be used for screening drug candidate molecules (drug target). It is useful, and can be used for, for example, a screening method for a substance that regulates the cell growth inhibitory effect of SPHK2 protein. Substances that regulate the cell growth inhibitory effect of SPHK2 protein include, for example, substances that inhibit the binding of SPHK2 protein to the substrate sphingosine and inhibit the cell growth inhibitory effect of SPHK2 protein, or conversely, SPHK2 protein And the like, which enhance the cell growth inhibitory action of the lipase. Such a substance can be used for diseases requiring cell proliferation, for example, regenerative medicine such as regeneration of skin and cornea, and a screening method thereof is also included in the present invention.

As the screening method of the present invention, various conventionally known methods for examining the presence or absence of bonding or dissociation between substances can be applied, and are not particularly limited. For example, by expressing the binding region of the SPHK2 protein with the substrate sphingosine, and in a cell-free system, a molecule that inhibits the binding between the binding region and the substrate sphingosine is proposed as a candidate. A screening method of detecting from molecules by an ELISA method or the like may be used.

 Thus, in the screening method of the present invention, in addition to the full-length protein of SPHK2, a partial protein thereof may be used. Further, a variant of the above full-length protein or partial protein may be used. As the above partial protein, (1) a binding region to substrate sphingosine, (2) a catalytic region (kinase active region), or (3) a region containing an NLS sequence can be used. In addition, the above “modified form of (protein)” means that one or several (preferably 7 or less, more preferably 5 or less, more preferably 3 or less) amino acids of the protein are replaced, Deletion, insertion, and Z or added variants, when the protein is labeled with a tag such as His or Myc, or when the protein is fused with a fluorescent protein (such as GFP 'luciferase) or another protein The term is used to include the case where the protein is modified by phosphorylation or sugar chain binding. Of course, the screening method of the present invention is not limited to the above-described method, and the screening may be performed in a cell system using cultured cells or the like, instead of the screening in a cell-free system. .

In addition, (1) a method of immobilizing a partial protein of SPHK2 (for example, the regions (1) to (3) above) on a column and searching for a substance that binds to it, or (2) an immunoprecipitation-immunoplot method Various conventionally known methods for examining the presence or absence of binding or dissociation between substances, such as a method for searching for substances that inhibit the binding of SPHK2 protein to the substrate sphingosine, can be applied to the screening method of the present invention. It is. Further, the screening method of the present invention includes: For example, a gene encoding the above protein may be used. Further, the screening method of the present invention comprises: 1) a method of searching for a substance that inhibits the translocation of SPHK2 protein into the nucleus, or 2) a method of searching for a substance that controls the movement of the SPHK2 protein between the cytoplasm and the nucleus, And so on. Although the SPHK2 protein is mainly present in the nucleus, the presence of a small amount in the cytoplasm may indicate that the protein may move between the cytoplasm and the nucleus. If so, substances that control the movement of SPHK2 between the cytoplasm and the nucleus are useful as drug candidate molecules (drug target). Furthermore, in the screening method of the present invention, screening may be performed using proteins other than humans, for example, mouse homologs, rat homologs, and homologs of other organisms.

(4) Screening kit

 The present invention also includes a screening kit for performing the screening method described in the above section (3). This kit may be any kit that can carry out the above-described screening method, and the specific configuration thereof is not particularly limited. For example, the above-mentioned screening kit may contain the full-length or partial protein of sphingosine kinase 2, a modified version of the protein, or a gene encoding these proteins.

 According to this kit, the screening method described in the above section (3) can be carried out simply and reliably, and a substance that regulates the sphingosine kinase 2 cell growth inhibitory action can be searched for.

Hereinafter, an example will be shown along the attached drawings, and an embodiment of the present invention will be described. Five

This will be described in more detail. Of course, the present invention is not limited to the following examples, and it goes without saying that various aspects can be provided in detail. It should be noted that the specific embodiments or examples made in the section of the best mode for carrying out the invention only clarify the technical contents of the present invention, and are limited only to such specific examples. It should not be construed as limiting in a narrow sense, but can be implemented with various modifications within the spirit of the present invention and the claims described below.

〔Example〕

 Hereinafter, results of a functional analysis performed on SPHK2, which forms the basis of the present invention, will be described with reference to the drawings.

[Example 1: SPHK2 is mainly localized in the nucleus]

 To examine the intracellular localization of SPHK2, a fusion protein (SPHK2-GFP) obtained by fusing mouse-derived SPHK2 with green fluorescent protein (GFP) was transferred to COS 7 cells using an expression vector. It was expressed transiently and observed with a confocal microscope two days later. The results are shown in Panel A of Figure 1. The par in the figure indicates a length of 10 z m (the same applies hereinafter). As shown in the figure, the SPHK2-GFP fusion protein was mainly present in the nucleus, and was hardly present in the cytoplasm.

 Nuclear localization of SPHK2 was also observed when transiently expressed in HeLa cells (see panel B in Figure 1). Similarly, when expressed in NIH 3T3 cells, nuclear localization of SPHK2 was observed (data not shown).

It was also possible that SPHK2 was localized in the nucleus by fusion with GFP. Next, a fusion protein of SPHK1 and GFP, which is an isozyme of SPHK2, was expressed, and its intracellular distribution was compared with that of SPHK2. The results are shown in panel C of FIG. As shown in the figure, the SPHK1-GFP fusion protein was mainly present in the cytoplasm, but not in the nucleus.

 Furthermore, instead of GFP, SPHK2 protein with HA tag added (HA-SPHK2) was expressed, and its localization was examined using an anti-HA antibody. The results are shown in Panel D of Figure 1. The HA-SPHK2 protein was mainly distributed in the nucleus as indicated by “S” in the figure, and its expression in the cytoplasm was slight. At the same time, nucleoporin staining was also performed, and the staining pattern clearly indicated the outer edge of the nucleus as indicated by “N” in the figure. The above results indicated that SPHK2 mainly exists in the nucleus.

 [Example 2: The nuclear translocation signal of SPHK2 exists in the region on the amino end side],

 SPHK2 (mSPHK2) derived from mouse is replaced by SPHK1 (mSPH2) derived from mouse.

It has 23.6 more amino acid residues than K1) (see the schematic diagram in Fig. 2 (a)). As shown in the figure, SPHK2 contains two long sequence regions that are not found in SPHK1 (one is at the amino end, the other is near the center).

The difference in intracellular localization between SPHK2 and SPHK1 was thought to be due to either of these two long sequence regions present only in SPHK2. There, two deletion mutants were created. One is mSPHK2 AM, which is deleted near the center, and the other is mSPHK2 AN, which is deleted at the amino terminal. When the mSPHK2ΔM-GP fusion protein was transiently expressed in COS 7 cells, the fusion protein was localized mainly in the nucleus (see Fig. 2 (b)). This result indicates that the center of SPHK2 does not affect the nuclear localization of the protein. On the other hand, when the mSPHK2 AN-GFP fusion protein was transiently expressed in COS 7 cells, this fusion protein could not translocate to the nucleus as shown in the figure. The above results indicate that the nuclear localization signal (NLS) is located in the amino-terminal region of SPHK2.

 [Example 3: Identification of nuclear localization signal (NLS) sequence in SPHK2]

Although the existence of the NLS sequence in the amino acid sequence of SPHK2 has not been reported before, this analysis identified the NLS sequence of SPHK2 derived from mouse. The NLS sequence is an arginine (R) -rich "RGRRGGRREJ sequence. As shown in Fig. 3 (a), (1) Tat of human immunodeficiency virus-1 (hum an immunodeiicie ncy virus-1) Similar to the NLS sequences present in proteins No. and Rev, ② Human T-cell leukemia virus type 1 Rex protein, and ③ Atypical protein kinase C (PKCλ). The consensus sequence of the NLS sequence (Consensus NLS) is also shown In the figure, each number indicates the position of the amino acid residue In the case of SPHK2, the first and last arginines are 8 in the full-length amino acid sequence, respectively. This indicates that they are located at positions 7 and 95. Bold letters indicate arginine and lysine, which are important for NLS function, and each sequence is aligned based on these amino acid residues.

To examine whether the above NLS sequence of SPHK2 really functions as an NLS, mutant SPHK2R93E / R94E-GFP was prepared. This mutation In the body, arginines 93 and 94 of the SPHK2-GFP fusion protein (arginine with an asterisk in Fig. 3 (a)) were both changed to glutamic acid (E). This mutant SPHK2R93E / R 94 £ -0? Was transiently expressed in 0807 cells, and two days after transfection of the expression vector, the cells were fixed and observed with a confocal microscope.As shown in Fig. 3 (b), this mutant protein was translocated to the nucleus. could not. To this end, a fusion protein in which the above NLS sequence of SPHK2 was fused with SPHK1, which is originally present in the cytoplasm, was expressed, and the presence or absence of its nuclear translocation was examined. That is, the fusion protein NLS-SPHK1-GFP was transiently expressed in COS 7 cells, and two days after transfection of the expression vector, the cells were fixed and observed with a confocal microscope, as shown in Figure 3 (b). This fusion protein mainly accumulated in the nucleus. The above results indicate that the NLS sequence is necessary and sufficient for nuclear translocation of SPHK2.

[Example 4: SPHK2 suppresses intracellular DNA synthesis] As described above, SPHK2 is localized in the nucleus, and it is thought that SPHK2 may be involved in the action in the nucleus. Was done. Therefore, changes in intracellular DNA synthesis were examined by measuring the incorporation of [ ³ H] thymidine into HeLa cells that stably express SPHK2. In experiments, we used doxycycline (dox) to induce the expression of SPHK2 in HeLa Tet-On cells into which SPHK2 had been introduced, and used these cells to induce various Dox concentrations (0 to lg / nil). ), The change in intracellular DNA synthesis under each concentration condition was examined using [ ³ H] thymidine incorporation as an index. The results are shown in the graph of Fig. 4 (a). Each value is the mean standard deviation of three independent experiments. As shown in the figure, cells in which the expression of SPHK2 was induced at a Dox concentration of 0.1 g / ml were compared with cells that did not express exogenous SPHK2 under the conditions without Dox (0 μg / ml). The uptake of [ ³ H] thymidine was suppressed by about 50%. Induction of SPHK2 expression by Dox was confirmed by immunoplot analysis. That is, HeLa Tet-On cells into which HA-SPHK2 had been introduced were placed under various Dox concentration conditions, and then subjected to SDS-PAGE followed by immunoblotting analysis using an anti-HA antibody. The result is shown in Fig. 4 (b). In addition, in the cells transfected with the expression vector without the SPHK2 gene, the Dox concentration had almost no effect on the incorporation of [ ³ H] thymidine (data not shown). The above results indicate that SPHK2 suppressed intracellular DNA synthesis.

 [Example 5: Nuclear localization of SPHK2 is essential for its DNA synthesis inhibitory activity]

 The mechanism of DNA synthesis suppression by SPHK2 is based on BrdU (bromodeoxyuridine

) Was also studied by measuring uptake.

 In the experiments, NIH 3T3 cells were used for (1) control HA-GFP, (2) HA-SPHK2-GI, (3) HA-SPHK2R93E / R94E.GFP, © HA-SPHK1-GFP, and (4) NLS-HA-SPHK1. An expression vector encoding any of -GFP was introduced, and two days after the introduction, BrdU was added to the cultured cells into which each vector had been introduced for 3 hours. Then, for each cell, the uptake of BrdU and all cells was visualized by double immunofluorescence, and the proportion of cells that had taken up BrdU among all the cells was determined. The results are shown in the graph of FIG. Each value is the mean of three independent experiments, the standard deviation of the soil.

As shown in the figure, when SPHK2 was expressed in NIH 3T3 cells, BrdU Uptake was significantly suppressed compared to cells transfected with the control vector. This result indicates that SPHK2 suppressed intracellular DNA synthesis. In contrast, cells expressing SPHK1 increased BrdU incorporation by about 40%.

 In addition, when the above-mentioned NLS sequence was modified and the mutant SPHK 2R93E / R94E, which could not be translocated into the nucleus, was expressed in cells, SPHK2 lost its DNA synthesis inhibitory activity as shown in the figure. Furthermore, fusion with the NLS sequence derived from SPHK2 and expression of the mutant NLS-SPHK1, which has nuclear translocation ability to SPHK1, resulted in suppression of DNA synthesis as shown in the figure.

 The above results indicate that nuclear localization of SPHK2 is essential for its DNA synthesis inhibitory action.

 [Example 6: SPHK2 expression does not induce apoptosis]

 When HeLa Tet-On cells transfected with SPHK2 were induced by Dox, SPHK2 was mainly expressed in the cell nucleus as shown in Fig. 6 (a) (comparison between Dox (-) and Dox (+). (Dox (+) refers to cells treated for 3 days under 0.1 μg / ml Dox conditions, and Dox (-) refers to cells treated for 3 days without Dox.)

Next, to examine whether apoptosis is induced in cells by SPHK2 expression, HeLa Tet-0n cells treated under the above-mentioned Ο.ΐ μg / ml Dox condition, and the above-mentioned Dox-free condition The DNA extracted from the HeLa Tet-On cells treated below was subjected to agarose gel electrophoresis, and its DNA pattern was examined. As a result, as shown in Fig. 6 (b), cells that induced SPHK2 expression under the above-mentioned Dox conditions (lane 1 in the figure) were induced to express under the above-mentioned conditions without Dox. Almost the same DNA pattern was obtained with the cells that were not induced (lane 2 in the figure). 'On the other hand, HeLa cells (lane 3 in the figure) treated with TNF-hi (tumor necrosis factor-) and cyclohexiraide, which are known to induce apoptosis, showed A typical apoptotic ladder pattern. These results indicate that SPHK2 suppresses intracellular ΝΑ synthesis but does not induce apoptosis.

[Example 7: Expression of SPHK2 causes cell cycle to remain in G1 / S phase] Next, the effect of SPHK2 on the cell cycle was examined. In the experiment, SPHK2-GFP expression vector (SPHK2-GFP expressed) or SPHK2-free vector (Mock treated) was expressed in 3-3 cells, and analyzed by flow cytometry. FIG. 7 (a) shows the results of the flow cytometry, in which the vertical axis represents the forward scattered light, indicating the size of the cell. The horizontal axis is the GFP fluorescence intensity. Double thymidine block initiates S phase in both cells with high GFP fluorescence (R2: ie, expressing SPHK2-GFP) and cells with low GFP fluorescence (R1: ie, not expressing SPHK2) Tuned to. Thereafter, the thymidine block was removed, and the DNA content (DNA content) of both R1 and R2 cells was analyzed at predetermined time intervals (0h, 3h, 9h) by flow cytometry using PI (propidium iodide). The result is shown in Fig. 7 (b).

Each histogram in the figure also shows the respective percentages (%) of the G1, S, and G2 / M phases, which are the measurement results. As shown in the figure, both R1 and R2 cells were synchronized at the beginning of S phase by double thymidine block. As a result, at 0 hours (O h) after removing the block, almost 100% of SPHK2-expressing cells (R 2) and non-expressing cells (R 1) did not enter the G2 / M phase.

 At 3 hours (3 h) after removal of the block, differences were observed between SPHK2-expressing cells (R2) and non-expressing cells (R1). After 3 hours in non-expressing cells (R 1), the majority of cells had entered S phase. At 9 hours (9 h) after the removal of the block, non-expressing cells (R 1) were divided into those that remained in S phase and those that transitioned to G 2 / M phase.

 In contrast, SPHK2-expressing cells (R 2) showed little change in the proportions at each stage after 3 hours, many of which remained in G1 phase and arrested at typical G1 / S phase showed that. At 9 hours (9 h) after block removal, about 40% of non-expressing cells (R1) entered the G2 / M phase, whereas SPHK2-expressing cells (R2) showed G2M. Only 7% were in the / M phase.

 These results indicate that expression of SPHK2 prolongs the cell cycle in the G1 or S phase and suppresses DNA synthesis.

 (Example 8)

Subsequently, in order to analyze the intracellular localization of SPHK2 in more detail, in addition to the results shown in Example 1, the localization of SPHK2 and its mutants was examined. Specifically, SPHK2, SPHK1, and their mutants were transiently expressed in COS7 cells, HeLa cells, or NIH 3T3 cells, and their intracellular localization was examined. Panels A and B in FIG. 8 correspond to panels A and B in FIG. 1, respectively. Panels and K in FIG. 8 correspond to the panels in FIG. 2 (b), and panels L and M in FIG. Since they correspond to the diagrams in FIG. 3 (b), the description is omitted here. A fusion protein (mSPHK2-GPT) obtained by fusing mouse-derived SPHK2 with green fluorescent protein GFP (Green Fluorescent Protein) was transiently expressed in NIH 3T3 cells using an expression vector. Observation was performed using a focusing microscope. The results are shown in panel C of FIG. The bar in the figure indicates the length of Ι Ο μ πι (the same applies hereinafter). As shown in the figure, the mSPHK2-GFP fusion protein was mainly present in the nucleus and hardly existed in the cytoplasm.

 In addition, it was examined whether there was a difference in intracellular localization due to the presence or absence of fusion with GFP or a difference in species. Specifically, first, human-derived SPHK2 (hS PHK2) was transiently expressed in HeLa cells, and intracellular intracellular by confocal microscopy using a specific antibody against the common peptide sequence of hSPHK2 and mSPHK2. Localization was observed. Panel D shows the results.

 As shown in the figure, hSPHK2 was found to be mainly localized in the nucleus, similar to mSPHK2. When HeLa cells were stained with the nuclear-specific stain 4, 6-dia midino-2-phenylindole (DAPI) (Fig. 8, panel E), it was confirmed that both DAPI and hSPHK2 were localized in the nucleus. (Panel F in Figure 8).

Next, a fusion protein, mSPHKl-GFP, was prepared by fusing SPHK1 (mouse-derived), SPHK's isozyme, with GFP. The intracellular localization of this mSPHKl'GFP was examined and compared with that of SPHK2. As shown in FIG. 8, panel H, in COS7 cells, mSPHKl-GFP was localized mainly in the cytoplasm and hardly localized in the nucleus. This is similar to previous reports (Olivera, A., Kohama, T., Edsall, L., Nava, V., Cuvillier, 0., Poulton, S., and Spiegel, S. (1999). ) J. Cell Biol. 147, 545-558). (Example 9)

Next, we examined whether the distribution of SPHK2 to the nucleus or cytoplasm is affected by cell type and cell confluency. Specifically, high density hSPHK2 (high: 3 X l 04 / mm2) or low density ^{(1 ow: 6 X 10 3} / mm2) expressing various cell lines of the state at (cell line) transiently I let it. Two days later, the transformed cells were fixed, immunostained with an anti-hSPHK2 antibody, and observed with a confocal microscope.

 Depending on the results of hSPHK2 staining, cells expressing hSPHK2 were divided into groups in which hSPHK2 was mainly localized in the nucleus (N> C), and groups in which hSPHK2 was localized almost equally in the nucleus and cytoplasm. (N = C), hSPHK2 force S was mainly divided into cytoplasm-localized group (N <C), and divided into three groups. Figure 9 shows the results.

 As shown in the table in FIG. 9, hSPHK2 transiently expressed in HeLa cells was found to be localized mainly in the nucleus under all conditions. It was found that hSPHK2 expressed in COS7 cells dramatically changed the intracellular localization depending on the cell density. In other words, it is mainly localized in the nucleus under high-density conditions (61.4%), but only 10.3% under low-density conditions. In contrast, hSPHK2 expressed in HEK293 cells was found to be localized mainly in the cytoplasm regardless of cell density.

 (Example 10)

Subsequently, it was examined whether or not the endogenous SPHK2 was localized in the nucleus as well as the exogenously expressed recombinant SPHK2. To address this issue, we used an anti-hSPHK2 antibody to immunoprecipitate endogenous SPHK2 from nuclear extracts of HeLa cells and analyzed its activity. Specifically, the procedure was performed as follows. First, in the presence / absence of 15 g / ml of the immunogenic peptide, the endogenous SPHK2 of the purified nuclear extract fraction was immunoprecipitated using an anti-hSPHK2 antibody. The sedimented precipitate was washed and SPHK2 activity was measured. The endogenous SPHK2 was 1.4 pmol SPP / min / tube. The results are shown in Fig. 10 (a).

 As shown in the figure, no band corresponding to SPP was found in the immunoprecipitate prepared in the presence of the immunogenic peptide, Byone, but clearly in the immunoprecipitate prepared in the absence of the immunogenic peptide. A band equivalent to the SPP was detected. This indicates that the purified nuclear extract fraction that specifically bound to the anti-SPHK2 antibody had SPHK2 activity.

 Next, fractionation of the immunoprecipitate prepared in the absence of the immunogen peptide was further analyzed by an immunization plot using an anti-hSPHK2 antibody. Specifically, immunoprecipitated immunoprecipitates without immunogenic peptides, affinity-purified HA-SPH Kl, and HA-SPHK2 were electrophoresed on 12.5% SDS-PAGE, and then anti-hSPHK2 antibody. Alternatively, immunoblotting was performed with an anti-SPHK1 antibody (Abeam, Cambridge, UK). The results are shown in Figure 10 (b).

As shown in the figure, a clear band was detected around 70 kDa in the sediment of the nuclear fraction. This corresponds to the position of affinity purified recombinant HA-hSPH K2. This approximately 70 kDa band is often detected as a double band, indicating the presence of a post-transcriptionally modified protein. The anti-hSPHK2 antibody did not crossreact with HA-SPHK1. Immunization of the precipitate with an anti-SPHK1 antibody did not produce immunoreactive bands around formal HA-SPHK1 or HA-SPHK2. These results strongly suggest that the anti-hSPHK2 antibody specifically recognizes endogenous SPHK2, but not SPHK1, and that the purified nuclear fraction contains endogenous SPHK2. Was. This result was clearly confirmed by immunocytochemical analysis.

 Specifically, HeLa cells were fixed and stained with an anti-hSPHK2 antibody, and observed with a confocal microscope. The nuclei were stained with 2 ^ g / ml DAPI. The results are shown in Fig. 10 (c). The bar in the figure indicates 10 μm. As a result, as shown in the figure, it was found that endogenous SPHK2 was localized mainly in the nucleus and slightly present in the cytoplasm. These results are similar to those of the overexpressed recombinant SPHK2 shown in Figures 1, 8, and 9.

 (Example 11)

 Subsequently, protein expression and enzyme activity of SPHK and various mutants were examined. Specifically, COS7 cells were transiently transfected with hSPHK2, HA-mS PHK1, NLS-HA-mSPHKl, or the plasmid vector pCMV5 and transformed. Two days later, the transformed cells were subjected to SDS-PAGE, and then subjected to immunoblotting with an anti-HA antibody or an anti-SPHK2 antibody. The results are shown in the inset of Fig. 11 (a). In addition, the enzymatic activity was measured using the fractionated cells. The results are shown as a graph in Fig. 11 (a).

As shown in the figure, COS7 cells transformed with only one vector had low SPHK activity. On the other hand, cells transformed with HA-mSPHKl, NLS-HA-mSPHKl, or hSPHK2 have 63-fold, 53-fold, or 5-fold higher in vitro SPHK activity than those transformed with vector alone. 2x rise did.

 In addition, the purified intact nuclear fraction was used to examine the accumulation of SPP in the nucleus in vitro. Specifically, HeLa cells were transiently transformed with hSPHK2, HA-mSPHK1, NLS-HA-mSPHKl, or the plasmid vector pCMV5. Two days later, the transformed cells were disrupted and fractionated. For the cytoplasmic fraction (C) and the nuclear fraction (N), SPP accumulation or LDH activity was measured. The results are shown in Fig. 11 (b).

 As shown in the figure, in the cells expressing hSPHK2, SPP mainly accumulated in the nuclear fraction (75%), but this result was similar to the morphological measurement shown in the table of FIG. Was. On the other hand, in cells expressing HA-mSPHKl, previous reports (Olivera, A., Kohama, T., Edsall, L., Nava, V., Cuvillier, O., Poulton, S., and Spiegel, S. (1999) J. Cell Biol. 147, 545-558), SPP accumulated mainly in the cytoplasmic fraction. In addition, when NLS-SPHK1 was expressed, the accumulation of SPP was clearly shifted from the cytoplasmic fraction to the nuclear fraction. LDH, a typical cytoplasmic marker protein, is hardly detected in the nuclear fraction. In addition, nucleoporin, a typical nuclear membrane marker, is detected in the nuclear fraction (data not shown).

 (Example 12)

Next, the intracellular distribution of the HA-SPHK2 protein was examined using different epitope-specific antibodies. Specifically, the SPHK2 gene (HA-SPHK2) with the HA tag attached to the N-terminal was introduced into COS7 cells, and one day later, immunostaining was performed using an anti-HA antibody or anti-SPHK2 antibody, and confocal laser microscopy. Observed using The results are shown in FIG. As shown in panels A to C in the same figure, when stained with anti-HA antibody, HA-SPHK2 protein is mainly distributed in the cytoplasm in the form of granules or directly below the cell membrane, and the nucleus is hardly stained. When stained, the cells were most densely distributed in the nucleus, and in the cytoplasm, a distribution image similar to that stained with the anti-HA antibody was observed.

 This strongly suggested that HA-SPHK2 translocated to the nucleus had its N-terminal HA tag-containing portion cleaved, and its antigenicity to anti-HA antibody was lost. Incidentally, since the recognition site of the anti-SPHK2 antibody is located in the center of SPHK2, it is presumed that the antigenicity is maintained.

Industrial potential

 As described above, the present invention relates to a cell growth inhibitor using sphingosine kinase 2, a method for producing a fusion protein having a nuclear translocation signal thereof, a method for screening a drug candidate substance, and the like. As described above, various cell culture techniques, regenerative medicine represented by the preparation of tissues such as skin tissue and the preparation of organs and organs, use in experimental systems using model animals, and cell proliferative diseases It can be used as a diagnostic or therapeutic agent for various diseases, and has various usefulness.

Claims

The scope of the claims 1. A cell growth inhibitor containing a mammalian sphingosine kinase 2 protein or a gene encoding the protein. 2. The cell growth inhibitor according to claim 1, wherein a human or mouse derived sphingosine kinase 2 is used. 3. Oligonucleotide that encodes a nuclear translocation signal at the amino terminus of mammalian sphingosine kinase 2. 4. A method for producing a fusion protein by using the oligonucleotide of claim 3 to fuse a nuclear translocation signal of sphingosine kinase 2 with another protein. 5. A fusion protein produced by the method according to claim 4. 6. The fusion protein according to claim 5, wherein a nuclear translocation signal of sphingosine kinase 2 and sphingosine kinase 1 are fused. 7. A cell growth inhibitor comprising the fusion protein according to claim 6, or a gene encoding the fusion protein. 8. A method for screening a substance that regulates the cell growth inhibitory effect of sphingosine kinase 2. 9. The screening method according to claim 8, wherein a full-length or partial protein of sphingosine kinase ₂ , a modified protein thereof, or a gene encoding these proteins is used. A screening method characterized by the following.  10. A drug containing a substance obtained by the screening method according to claim 8 or 9.  11. A screen jungle kit for performing the screen jungle method according to claim 8. 12. The scope of the claim, wherein the above-mentioned screening kit includes a full-length or partial protein of sphingosine kinase 2, a modified form of the protein, or a gene encoding these proteins. 11 Screening kit described in 1.