KR100372180B1 - A novel Drosophila MAP Kinase Phosphatase which specifically inhibit ERK - Google Patents

Abstract

The present invention relates to a novel gene that specifically inhibits tumors caused by abnormal signal transduction of the map kinase signal pathway, and the present invention relates to a Drosophila dual specific map kinase dephosphatase that specifically inhibits ERK map kinase enzyme. The gene of the present invention can be used as a new and effective anticancer agent that can act on most tumors caused by abnormality of ERK regulation.

Description

A novel Drosophila MAP Kinase Phosphatase which specifically inhibits ERK}

The present invention relates to a novel gene that specifically inhibits the development of tumors caused by abnormal activation of the map kinase signal pathway, and more particularly, a new map kinase dephosphatase of Drosophila (hereinafter, referred to as ERK) that specifically inhibits ERK. Invention).

Recently, many studies have been conducted on the cell signaling pathways that induce the development and progression of various types of tumors. In particular, many studies are focused on identifying mutated genes and proteins, as well as abnormalities in cancer cells compared to normal cells. Genetic studies have revealed gene families with mutations that can produce many types of tumors.

MAP kinase pathways are important cellular networks in which cells induce cell growth, differentiation and epoptosis in response to multiple extracellular stimuli, such as growth factors, nutritional status, and stress, and their signaling between eukaryotes from yeast to humans The mechanism is well preserved. To date, three important map kinase signal pathways such as ERK, SAPK, and p38 are well known, and balanced signaling between these map kinase pathways is an important factor in determining cell fate and homeostasis. In this case, it has been found to be closely associated with the development of many types of tumors.

Anticancer agents commonly used in the treatment of tumors associated with the map kinase pathway, such as inhibitors of panesyltransferase and antibodies that inhibit receptor tyrosine kinase, which act at an upper stage of the map kinase signal pathway, as in US Pat. No. 5,420,245. Anticancer drugs are common. However, the anticancer agents that inhibit the MAP kinase signal pathway in the upper stages had many problems such as side effects caused by the "cross-talk" that may occur in the lower stages of the MAP kinase signal pathways, and decreased in effectiveness.

The present invention has been made to solve the above problems, and an object of the present invention is to isolate a gene that specifically inhibits ERK, which is the lower stage of the MAP kinase signal pathway, and to identify a tumor resulting from abnormal ERK regulation. It is to provide as a specific anti-cancer gene.

DMKP, which acts specifically on the ERK of Drosophila newly discovered in the present invention, is well preserved in a variety of higher organisms ranging from yeast to human, and does not inhibit JNK or p38 MAPKs. Can be developed as an anticancer agent.

1 is a figure measuring the amount of Drosophila MAP kinase phosphatase (DMKP) mRNA during the development of Drosophila using RT-PCR.

Figure 2 A is a diagram showing the expression of His-DMKP in E. coli, B is the expression of DMKP in yeast, C is a figure confirming their activity.

Figure 3 shows the results of expressing DMKP using CuSO ₄ in a cell line into which the DMKP gene was stably inserted.

4 is a diagram showing that DMKP is a dephosphatase that specifically acts on ERK among ERK, JNK, and p38 MAP kinase. When AK is expressed in a cell line in which DMKP gene is stably inserted, ERK (A), JNK (B) and p38 (C) MAPK activity is shown respectively.

Figure 5 shows that when the DMKP gene is expressed in S. cerevisiae , it inhibits Fus3 dependent gene expression by inhibiting Fus3 MAPK similar to ERK.

Figure 6 shows that DMPK inhibits the activity of FUS3 MAP kinase.

In order to achieve the above object, the present invention provides a novel map kinase dephosphatase gene of Drosophila that specifically inhibits ERK.

The present invention also provides an expression vector for expressing the gene.

The expression vector is preferably at least one expression vector selected from the group consisting of pBS-DMKP, pKC305, pPac-DMKP and pKC423.

The present invention also provides a novel map kinase dephosphate enzyme of Drosophila described in SEQ ID NO: 2 expressed by the above gene.

Hereinafter, the present invention will be described in detail.

The activity of mapkanase requires phosphorylation of threoine and tyrosine residues of the conserved Thr-X-Tyr motif. Phosphorylation of MAPKinase is a reversible reaction mediated by MKPs or MKPs that are mostly bispecific or MKPase specific kinase dephosphatase. Genes involved in MAP kinase signaling are well preserved from vertebrates to yeast. The mechanism of inactivation of map kinase is also preserved across species, as is the mechanism of map kinase activation. Various regulation of MAP kinase by MAP kinase dephosphatase is very important for cross regulation between various MAP kinase signal pathways, and specific inhibition of MAP kinase has also been reported. MAPPanase Dephosphatase (MKP) -3 is highly specific for ERK and has recently been shown to not inactivate JNK or p38 MAPKinase and is being developed as an important next-generation anticancer agent.

In Drosophila , as in mammals, three other classes of map kinase have been identified. Drosophila Rolled / ERKA (DERK), coded by rolled, is a component of the Sevenless signaling warning, which regulates various differentiation and development processes. DJNK, the JNK homolog of Drosophila , is important for the morphological process of dorsal sutures, immune responses, photoreceptor determination and formation of tissue polarity. Drosophila p38 (Dp38) has also been recently cloned. However, Drosophila 's MAP kinase dephosphatase gene, which is involved in the regulation of MAP kinase, has not yet been identified except for Puckered, which specifically inhibits JNK. Activation of MAP kinase is a transient one that must be regulated, and failure to inactivate MAP kinase can lead to the transition from higher eukaryotic cells to carcinogenesis.

Thus, the present inventors searched for Drosophila- expressed sequence tag data (hereinafter referred to as "dbEST") and described a novel Drosophila MAPKinase Dephosphatase (DMKP) cDNA with similarity of amino acid sequence to MAPKinase dephosphatase with known bispecificity. The enzyme was identified as a dephosphatase that specifically acts on ERK MAP kinase, and was used for the treatment of tumors resulting from abnormalities in the MAP kinase pathway by expression or injection at the protein level.

Restriction enzymes used in the present invention were purchased from New England Biolabs Inc., Schneider medium, fetal bovine serum, antibiotics, lipofectamine was purchased from Life Technologies, Inc. Tri reagents for RNA isolation are described in Molecular Research Center Inc. 6x His tag plasmid, pQE31, and QIAexpress Ni-NTA protein purification system were purchased from Qiagen Inc. Purchased from PMT / V5 and pCoHygro vectors were prepared from Invitrogen Co. to prepare stable cell lines. The PacPL vector used to prepare PacPLDMKP with Drosophila actin promoter was supplied by Dr. D. Huntmark of Umea University, Sweden. The minimum SD medium for complete yeast synthesis medium was purchased from Clontech Laboratories, Inc. The ECL system was developed by Genepia Inc., Seoul, Korea. The high sensitivity X ray film was purchased from Amersham Inc. p-ERK, -JNK and -p38 antibodies were purchased from New England Bio Lab. Monoclonal anti-α tubulin antibodies are described in Upstate Biotechnology Inc. Purchased from Research Product. Anti-rabbit IgG antibody and protein assay kits were purchased from Bio-Rad Laboratories and protran nitrocellulose membranes from Schleicher and Schuell Co. All other chemicals, including para-nitrophenyl phosphate (hereinafter referred to as "pNPP"), protein A sepharose, amino acids and protease inhibitors, were purchased from Sigma.

Hereinafter, the present invention will be described in more detail with reference to non-limiting examples.

Example 1

In order to know the new Drosophila MKP, the inventors find a Drosophila sex dbEST for amino acid sequence homology to one of the dual-specific MKP, MSG5 commanded la DMKP it. This cDNA also has a size of 1.4-kb, a 3'-poly A structure, and a 5'-untranslated region. DMKP ORF is a polypeptide of 203 amino acids with HCXXGXXRS / T sequences well conserved at the activation site. DMKP also has Asp-120, Cys-152, and Ser-159 residues conserved in the catalytic site of phosphatase with bispecificity, and also has a GYLM motif.

These results were obtained through the following experiment.

By investigating Drosophila dbEST with a dual specific mapkinase dephosphatase (hereinafter referred to as "MKP") amino acid sequence, the inventors of the present invention have found that partial cDNA (accession) has a large homology with the C-terminus of MKP. No. CK00185) was confirmed. The cDNA clone in plasmid (CK00185), pBS-DMKP-CK185, was obtained from the Drosophila genome resource at the University of California, USA. The base sequence of total cDNA was performed by the Sanger method (Proc. Natl. Acad. Sci. USA 74 (1977) 5463-5467). The MacVector ^™ 6.0.1 program was used for amino acid sequencing.

[Production of plasmid]

The vector sequence of pBS-dMKP-CK185 located at the 3 'end of Drosophila map kinase dephosphatase (hereinafter referred to as "DMKP") was removed by treatment with Nde I and Xba I. PBS-DMKP and pKC305 were prepared by relinking the plasmids after filling the ends with dNTP and Klenow fragments. pPac-DMKP was prepared by inserting the Eco RV and Sac Ⅰ fragment of pBS-DMKP into the Eco RV and Sac Ⅰ seat of PacPL. pBS-DMKP, E. coli expression vectors of the C- terminal domain of pKC379 is 6x His tag plasmid, pQE31 of BamH Ⅰ and Sac Ⅰ position in the BamH Ⅰ of pPac-DMKP having the cDNA for the 154 amino acids of the C- terminal And Sac I fragments were prepared. To prepare a yeast DMKP expression vector (pKC423), total DMKP cDNA by PCR of pKC305 (pBS-DMKP) with two primers with Hind III sites, namely 5'-ACCGAGAAACGAAGCTTTAATACCGCTATGAT-3 'and 5'-TTCATAAAAGCTTATCATATGCAGTATC-3' A 674 bp DNA fragment was obtained.

After agarose gel purification, the PCR product was subcloned to Hind III site of Ycp88 ( URA3 CEN ) to produce pKC423 .

Example 2: Drosophila Experimental expression of mRNA during development

We used RT-PCR to measure the level of DMKP mRNA during the development of D. melanogaster . As shown in FIG. 1, DMKP mRNA is not detected in the early embryo (0-4 hours) and stage 1 of larva, and detection of DMKP mRNA starts at stage 2 and becomes maximal at stage 3 and is maintained until adulthood. Expression of DMKP mRNA was also detected in l (2) mbn and Schneider cells. However, mRNA levels of β-actin did not change significantly throughout the developmental stage.

These results were obtained through the following experiment.

At several different developmental stages, total RNA from Drosophila melanogaster was extracted with Tri reagent according to the manual. The first strand of cDNA was synthesized in a reaction mixture with 5 μg total RNA, 1 dM of each dNTP, 20 units of AMV reverse transcriptase, and 1.6 μg random primer. 25 μl of reaction mixture has cDNA from 100 ng total RNA, 1.25 units of T. aquaticus DNA polymerase, 200 μM of each dNTP, 200 nM of each primer and 1.5 mM of MgCl ₂ .

The sense primer sequence of DMKP is 5'-CCCGCGTCAATGGAGTTTATTG-3 'and the antisense sequence is 5'-GATCACCATTGGCAACGA-3'. RT-PCR was denatured at 94 ° C for 30 seconds, annealed at 60 ° C for 1 minute, and at 1 ° C chain extension at 72 ° C with DNA thermal cycler of Perkin Elmer Co., β-actin amplification 20 times, DMKP amplification 30 times of each were performed. 10 μl of PCR product was separated and observed by 1.5% agarose gel electrophoresis with 0.5 ug / ml of Et-Br.

Example 3: Phosphate Activity Experiment of DMKP

To determine whether DMKP has phosphatase activity, the C-terminal domain of DMKP having the active site of E. coli as His-DMKP was expressed (see FIG. 2A). HIS-DMKP protein was dissolved in urea solution and purified to 99% purity. Purified DMKP was used to produce anti DMKP antibodies. Since DMKP protein produced in E. coli is insoluble, DMKP protein was produced in S. cerevisiae to detect phosphatase activity. The protein produced in yeast was 23 kDa. Immuno-purified DMKP hydrolyzed concentration dependently pNPP, a chromogenic substrate structurally associated with phospho-tyrosine (see FIG. 2C). However, no phosphatase activity was detected in the immunoprecipitates of extracts obtained from cells transformed with the vector alone.

These results were obtained through the following experiment.

[DMKP Expression and Antibody Production]

The C-terminal domain of DMKP (His-DMKP) tagged with 6x His sequence was expressed in bulk using E. coli BL21 (DE3) transformed with His-DMKP expression vector, pKC379. Cells were grown in LB medium with 50 μg / ml ampicillin. When the cell density reached the medium log phase, DMKP was induced with 0.2 mM isopropyl-1-thio-beta-D-galactopyranoside (IPTG). Five hours after induction, cells were collected and extraction was performed. Most recombinant His-DMKP proteins overexpressed in E. coli are insoluble. Insoluble His-DMKP protein was dissolved in 8 M urea and purified using a QIAexpress Ni-NTA protein purification system. DMKP antibody was obtained by injecting purified His-DMK protein into New Zealand white rabbit. The first immunization was carried out by mixing 500 μg of purified protein with 1 ml of Freund incomplete adjuvant, and 2 and 3 weeks with 200 ml and 300 μg of DMKP protein mixed with 1 ml of Freund complete adjuvant, respectively. Progress was made at intervals. One week after the third inoculation, serum was obtained and DMKP antibodies were purified by standard methods by Antibodies (1988), such as Halow et al.

DMKP expression in S. cerevisiae

pKC423 ( DMKP URA CEN ) or vector ( URA CEN ) were transformed into S. cerevisiae EY957 ( MATα sst1ΔKSS1 ura 3-1 leu2-3, 112trp1-1 his3-11,15 ade2-1 can1-100 + ), The transformants were selected from synthetic complete media lacking uracil. Yeast transformants were grown in synthetic fully selected medium, and extracts were made by the best method (JBC (1998) 273, 369-74).

Western Blot Analysis

Cell extracts with 50 μg of protein were separated by 10% SDS-PAGE and transferred to nitrocellulose membrane. Blots were blotted with 20 mM Tris-Cl, 150 mM NaCl and 0.05% Tween 20 (TBST) containing 5% skim carnation milk, washed three times with TBST and then with TBST milk with the appropriate amount of primary antibody. Incubate at room temperature for 2 hours or overnight at 4 ° C. The blot was then washed three times at 15 minute intervals and then incubated for 1 hour with TBST milk with Levit IgG. The membrane was washed three times with TBST at 15 minute intervals, and the blots were developed with ECL.

[Phosphatase Analysis]

200, 400 and 500 μg of yeast extracts obtained from DMKP transformants were immunoprecipitated using DMKP antibodies, and enzyme activity was measured for pNPP. The phosphatase reaction was carried out for 1 hour at 37 ° C. in a solution with 20 mM pNPP, 50 mM imidazole and 5 mM dicytotol. The reaction was stopped by the addition of 1 M NaOH and phosphatase activity was determined by measuring absorbance at 405 nm.

Example 4 Experimental Inhibition of DERK of DMKP in Drosophila Cells The present inventors examined whether DMKP can modulate the activities of DERK, DJNK and Dp38 MAPKinase dephosphatase in Schneider cells. Creating a stable Schneider cell line having DMKP gene whose expression is induced by the CuSO _4, DMKP protein was detected by DMKP antibody, CuSO ₄ induction of the chromosome in a cell having a DMKP gene, the amount the protein level as the increased CuSO ₄ dose-dependently Increased. The size of the DMKP protein observed on the gel was 29 kDa. Except for the CuSO ₄ inducing band, DMKP antibodies also recognized 33 kDa and 50 kDa proteins (see FIG. 3).

After successful induction of DMKP in stable cell lines, it was tested whether the expression of protein could inhibit the activity of DERK, DJNK and Dp38 MAPKinase dephosphatase. The level of DERK activity measured by phospho-DERK levels was very high in non-triggered cells, measuring the effect of DMKP in the regulation of DERK without stimulation. As shown in FIG. 4A, phospho-DERK levels are inversely related to DMKP levels induced by different CuSO ₄ ion concentrations. The levels of p-DJNK and -Dp38 can be increased significantly by treating cells with LPS or NaCl, respectively (see 4B or C), but the levels of phospho-DJNK and -Dp38 increased by promoters induce DMKP expression. Not significantly lowered (see 4B or C). Specific concentration dependent inhibition of phospho-ERK by DMKP was also observed in transient transfection experiments (see FIG. 5). Although p-DERK levels decreased gradually with increasing levels of transfected pPac-DMKP vector, the levels of phospho-DJNK and phospho-Dp38 were not changed by transfection of DERK expression vectors.

These results were obtained through the following experiment.

[Cell Culture and Preparation of Stable DMKP Cell Line]

Schneider cells were grown in 23 ° C. Schneider medium fed with heat inactivated 10% FBS, 100 units of penicillin and 100 ug / ml of streptomycin. Transient transformation was performed using lipofectamine. Stable cell lines expressing DMKP were prepared by transfection with pMT / V5-DMKP like pCoHygro with E. coli hygromycin-B-phosphortransase under the control of the Drosophila Copia promoter. Transfection was performed according to the standard CaPO ₄ protocol of Di Nocera, PP et al. (PNASUSA (1983) 80, 7095-98), and transfected cells were selected with 300 g / ml of hygromycin for 6 weeks. . Before use, stable cells were treated with 1 mM CuSO ₄ for 24 hours to induce DMKP expression. Induction of DMPK protein was analyzed by Western blot analysis using DMKP antibody. To investigate the activity status of Drosophila ERK (hereinafter referred to as "DERK"), total cell extracts from CuSO ₄ induced cells stably expressing DMKP were prepared, and Western-based anti-human phospho-ERK specific antibodies were used. Blot analysis was performed. To investigate the activity of cells DJNK and Dp38, CuSO ₄ induced cells expressing DMKP or uninduced cells stably, 10 ug / ml LPS or 300 uM NaCl for the activity of DJNK and Dp38, respectively. Treatment was for minutes.

[Preparation of Drosophila Cell Extracts]

Cultured cells were collected after washing twice with cold PBS. Cells were 300-500 ul 50-100 mM beta-glycererophosphate at pH 6.2- 8.2, 0.1-1 mM meta- and ortho- vanadate, 2 mM magnesium chloride, 1 mM EGTA, 1-3 mM Dithiothritol (DTT), 0.5% Triton X-100, 0.2-0.6 mM phenylmethylsulfonyl fluoride (PMSF), 5 μg / ml pepstatin A, chymostatin, Lysis was performed by dissolving in a cold buffer consisting of leupeptin and peptin. The sample was sonicated for 30 seconds after standing 15 minutes on ice. Unbroken cell debris was removed by centrifugation at 12,000 × g for 10 minutes and the supernatant was cleared by a second centrifugation. Samples immediately liquidified and frozen at -80 ° C for later use.

Example 5: Down regulation of the S. cerevisiae pheromone pathway of DMKP .

In order to know the function of DMKP in vivo, Elion et al. (P.N.A.S.U.S.A.88 (1991) 9392-9396) were used.

The fus1-lacZ reporter has a bacterial lacZ gene fused with a pheromone-induced promoter from the fus1 gene that is used to quantify the pheromone response. Expression of the fus1-lacZ reporter is dependent on the activity of FUS3 MAPKinase. Therefore, the present inventors measured the expression of FUS3- dependent fus1-lacZ trans-labeled gene in EY957, the sst1Δ strain, and tested whether DMKP inhibited the activity of FUS3 MAP kinase. Since SST1 is an enzyme that degrades the α factor, EY957 ( sst1Δ ) sensitively detects pheromone signaling. Fus1-lacZ gene expression increased 100-fold upon pheromone treatment (see FIG. 6). Β-galactosidase activity increased by pheromone was down regulated by about 25% in transformed with DMKP expressing plasmid compared to transformed with yeast vector only (see FIG. 6).

The above result was obtained by the following experiment.

[ fus1-lacZ trans-label system]

pKC423 ( DMKP CEN URA ) or empty vector ( CEN URA ) was transformed into EY957 with pJB207 ( fus1-lacZ 2μ LEU ) and selected from SC (selective complete) -Ura Leu plate. Inoculated in SC-Ura Leu medium and overnight at 37 ° C. Cells were grown by inoculating 1/10 volume of overnight culture and further grown on 30 ° C. shaking culture. When the OD ₆₀₀ value of the culture reached 1.0-1.2, the cells were induced for 1 hour with 50 nM α-factor, and extracts were made by the best method (Cell (1994) 78, 499-512).

[β-galactosidase analysis]

β-galactosidase analysis was performed by Dolan et al. (Genes Dev. (1990) 4, 492-502), and three independent transformants were used for β-galactosidase analysis to average the values. .

The present invention has found a new Drosophila bispecific mapkinase dephosphatase that is not significantly affected by the activity of DJNK and Dp38, and is involved in the specific down regulation of DERK, which is responsible for the regulation of the enzymes involved in the mapkinase pathway. The gene of the present invention can be used for the treatment of tumors by transfecting the genes of the present invention into tumors caused by inhibition of the regulation of ERK.

Claims (4)

A novel map kinase dephosphatase gene of Drosophila according to SEQ ID NO: 1 that specifically inhibits ERK. An expression vector selected from the group consisting of pBS-DMKP, pKC305, pPac-DMKP and pKC423 containing the gene of claim 1. delete A novel map kinase dephosphate enzyme of Drosophila according to SEQ ID NO: 2 expressed by the gene of claim 1.