KR100906153B1 - Protein for inhibiting PKC activity and pharmaceutical composition including the same - Google Patents

Abstract

The present invention relates to a protein capable of inhibiting the activity of PKC and a pharmaceutical composition comprising the same, more specifically, a nucleic acid molecule encoding a protein capable of inhibiting PKC activity, a vector comprising the nucleic acid molecule and Cells provided herein, the proteins and compositions of the present invention can prevent or treat tumors caused by PKC activity.

p47phox, PKC, cell carcinogen

Description

Protein for inhibiting PKC activity and pharmaceutical composition including the same

The present invention relates to proteins that inhibit PKC activity.

The present invention also relates to nucleic acid molecules encoding proteins that inhibit PKC activity.

The present invention also relates to a pharmaceutical composition for preventing or treating a tumor containing the protein.

The delivery system plays a major role in the pathogenesis of tumors so far identified, and molecular target anticancer drugs of various pathways have been developed based on this mechanism of action. Inhibitors that target tyrosine kinase (RA), Ras, protein kinase C (PKC), and mitogen-activated proteinin kinase (MAPK), all of which play a major role in tumor growth, all signal transduction in intracellular tumor development mechanisms. There is a high possibility that it can be developed as an anticancer drug. For example, the leukemia treatment drug “Gleevec” is an anticancer agent that blocks the tyrosine phosphorylation of the protein enzyme p120, which is involved in the carcinogenic mechanism of chronic myelogenous leukemia. EGFR (epidermal growth factor receptor) also corresponds to a tyrosine kinase that acts on a carcinogenic mechanism. Irressa specifically inhibits EGFR to block tumor growth and angiogenesis, thereby showing anticancer effects. Like p120 and EGFR, inhibitors to proteins such as Ras, MAPK, and PKC, which have a significant effect on tumor development, are also emerging as potential anticancer drugs.

Among these, PKC, which is found in many tissues and organs of animals, is a protein that appears in many tissues or organs of animals such as the brain, spleen, and heart. In general, PKC is known to have a function of signal transdution, cell growth, gene expression, and tumor promotor.

 In many animal cells, PKC is known to be involved in cell activity due to phosphorylation and pH change due to changes in Na + and H + concentrations, and in some cells it is known to be a signal of proliferation. PKC also increases the transcription of certain genes. In particular, the activity of PKC by DAG in secondary messengers produced by the hydrolysis of phosphatidylinositol can be similarly expressed by the binding of monoacylglycerol or phorbol ester. Phorbol esters have the function of a cancer promoter in animal cells and are known to induce the growth of cancer cells. These phorbol esters as well as oncogenic genes activate PKC and strongly activate the signaling system in cells involved in the growth of cancer cells.

PKC is also known to play an important role in the production of free radicals (ROS) in cells. The production of free radicals such as superoxide anions in cells is known to occur through the enzyme NADPH oxidase, xanthine oxidase, NO synthase, and mitochondrial electron transfer system. Among these various enzymes, NADPH oxidase consists of several subunits such as cytochrome b558, gp91phox, p22phox or Nox1 in the cell membrane, and p47phox, p67phox and small G protein Rac in the cytoplasm. Phosphorylation of p47phox or p67phox by signaling mechanisms in the cytoplasm moves to the cell membrane and binds to cytochrome b558. As a result, active oxygen (ROS) such as excess oxygen anion is generated from the activated NADPH oxidase.

It has been reported that the phosphorylation of p47phox, which is involved in the production of free radicals in cells, and the migration to cell membranes are regulated by direct binding of activated PKC.

In order to examine the effects of several domains of p47phox on the activity of PKC, in vitro and in vivo experiments showed that the sites corresponding to amino acid sequences 156-215 of p47phox were involved in the inhibition of PKC activity. When the peptide sequence corresponding to this site is administered to the cells, it is confirmed that the carcinogenic mechanisms induced by PKC activity may be inhibited and thus the effect of preventing or treating tumors may be exhibited. The present invention has been completed.

One object of the present invention is to provide a nucleic acid molecule encoding a protein that inhibits PKC activity.

Another object of the present invention is to provide a protein that inhibits PKC activity.

Another object of the invention to provide a pharmaceutical composition for the prevention or treatment of a tumor comprising a protein encoded by the nucleic acid molecule.

The present invention relates to a nucleic acid molecule encoding a protein capable of inhibiting PKC activity consisting of the amino acid sequence of SEQ ID NO: 2.

In the present invention, the nucleic acid molecule is DNA, preferably cDNA.

The present invention relates to a nucleic acid molecule encoding a protein capable of inhibiting PKC activity consisting of the nucleotide sequence of SEQ ID NO: 4.

The present invention relates to a recombinant vector comprising the nucleic acid molecule described above.

The present invention relates to a transformant transformed with an expression vector.

The present invention relates to a protein capable of inhibiting PKC activity encoded by the nucleic acid molecule.

The present invention relates to a protein capable of inhibiting PKC activity consisting of the amino acid sequence of SEQ ID NO: 2.

The present invention relates to a pharmaceutical composition for the prevention or treatment of a tumor, comprising a protein encoded by the nucleic acid molecule and a pharmaceutically acceptable carrier.

The present invention relates to a nucleic acid molecule encoding a protein capable of inhibiting PKC activity. The nucleic acid molecule inhibits the activity of PKC, thereby inhibiting the carcinogenic mechanism induced by PKC activity, and is effective in preventing or treating tumors.

In one embodiment, the present invention provides nucleic acid molecules encoding proteins that can inhibit PKC activity. The protein consists of the amino acid sequence of SEQ ID NO.

In the present invention, "PKC" is a protein kinase C. Looking specifically at the activation stage of PKC, signals outside the cell activate G-proteins through receptors of the plasma membrane and activate the G-proteins. G-proteins are chained to activate phospholipase C. In this series of processes, phosphatidylinositol is hydrolyzed, resulting in diacylglycerol (DAG) and inositol-1,4. Secondary transporters of, 5-triphosphate (inositol 1,4,5-triphosphate (IP3) are produced, which directly or indirectly affect cell activity, one of the second transporters, IP3, extracellula by causing the movement of Ca ⁺ ₂ into the plasma from the fluid), and increasing the concentration of Ca ₂ ⁺ in the cell, and the other of DAG activates protein kinase of particular PKC in the cells.

In the present invention a “protein”, “polypeptide” or “amino acid sequence” includes minor amino acid variations from natural amino acid sequences which include not only identical amino acid sequences but also isomers thereof and which include conservative amino acid substitutions. . Also included are amino acid sequences that produce polypeptides that are immunologically identical or similar to polypeptides that are mutated from naturally occurring amino acids but encoded by naturally occurring sequences. Preferably "amino acid sequence" in the present invention refers to the amino acid sequence of 156 to 215 of the p47phox protein, referred to as KIRAMS-NP47-SH3N.

The “nucleic acid sequence” of the present invention includes the sequence encoding SEQ ID NO: 4, its functional equivalents and functional derivatives.

In another aspect, the present invention relates to a recombinant vector comprising a nucleic acid sequence encoding the PKC activity inhibitory protein.

As used herein, a "recombinant vector" refers to a gene construct that is capable of expressing a protein of interest in a suitable host cell, and includes a gene construct that contains essential regulatory elements operably linked to express the gene insert.

As used herein, the term “operably linked” refers to a functional linkage of a nucleic acid expression control sequence and a nucleic acid sequence encoding a protein of interest to perform a general function. The promoter and the nucleic acid sequence encoding the protein may be operably linked to affect the expression of the encoding nucleic acid sequence.Operative linkage with the recombinant vector may be prepared using genetic recombination techniques well known in the art. Site-specific DNA cleavage and ligation uses enzymes generally known in the art.

Vectors of the invention include, but are not limited to, plasmid vectors, cosmid vectors, bacteriophage vectors, viral vectors, and the like. Suitable expression vectors include signal sequences or leader sequences for membrane targeting or secretion in addition to expression control elements such as promoters, operators, initiation codons, termination codons, polyadenylation signals and enhancers and can be prepared in various ways depending on the purpose. The promoter of the vector may be constitutive or inducible. The vector also includes a selection marker for selecting a host cell containing the vector and, in the case of a replicable vector, the origin of replication.

In another aspect, the present invention relates to a transformant transformed with the recombinant vector.

Transformation includes any method of introducing nucleic acids into an organism, cell, tissue or organ, and can be carried out by selecting appropriate standard techniques according to the host cell as known in the art. These methods include electroporation, plasma fusion, calcium phosphate (CaPO ₄ ) precipitation, calcium chloride (CaCl ₂ ) precipitation, agitation with silicon carbide fibers, agro bacterial mediated transformation, PEG, dextran sulfate, Lipofectamine and the like, but is not limited thereto.

Depending on the host cell, the expression level and the expression of the protein are different, so you can select and use the most suitable host cell for the purpose. Host cells include Escherichia coli, Bacillus subtilis, Streptomyces, Pseudomonas, Proteus mirabilis or Staphylococcus. Prokaryotic host cells such as, but are not limited to. In addition, fungi (eg Aspergillus), yeast (eg Pichia pastoris, Saccharomyces cerevisiae) Cells derived from higher eukaryotes, including lower eukaryotic cells such as Schizosaccharomyces and Neurospora crassa, insect cells, plant cells, mammals and the like, can be used as host cells.

In another aspect, the invention provides a composition for inhibiting PKC activity comprising a protein encoded by said nucleic acid molecule.

Increases in PKC activity include breast carcinoma, lung carcinoma, gastric carcinoma, glioblastoma, bladder carcinoma, colon carcinoma, prostate carcinoma, and myeloid leukemia. (myeloid leukemia).

Thus, the composition of the present invention is preferably a composition for preventing or treating tumors.

The composition containing the protein as an active ingredient may be used in the form of a general pharmaceutical formulation. Formulations for parenteral administration may include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, suppositories. As the non-aqueous solvent and the suspension solvent, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like can be used. As the base of the suppository, witepsol, macrogol, tween 61, cacao butter, laurin butter, glycerogelatin and the like can be used. In addition, the peptide may be used in admixture with a number of carriers (pharmaceutically acceptable) such as physiological saline or organic solvents, and carbohydrates such as glucose, sucrose or dextran to increase the stability or absorption of the inhibitor. Antioxidants such as ascorbic acid or glutathione, chelating agents, small molecule proteins or other stabilizers can be used as medicaments.

Administration is by the usual route of introducing the desired substance to the patient in a suitable manner. Intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, oral administration, topical administration, nasal administration, pulmonary administration, rectal administration, but is not limited thereto. However, upon oral administration, since the peptide is digested, it is desirable to formulate the oral composition to coat the active agent or to protect it from degradation in the stomach. In addition, the pharmaceutical composition may be administered by any device in which the active agent may migrate to the target cell. Preferred modes of administration and preparations are intravenous, subcutaneous, intradermal, intramuscular, injectable and the like. Injectables include non-aqueous solvents such as aqueous solvents such as physiological saline solution and ring gel solution, vegetable oils, higher fatty acid esters (e.g., oleic acid, etc.), and alcohols (e.g., ethanol, benzyl alcohol, propylene glycol, glycerin, etc.). Stabilizers (e.g. ascorbic acid, sodium bisulfite, sodium pyrosulfite, BHA, tocopherol, EDTA, etc.), emulsifiers, buffers for pH control, to prevent microbial development Preservatives such as mercury nitrate, chimerosal, benzalkonium chloride, phenol, cresol, benzyl alcohol, and the like.

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

Example 1: Preparation of GST Fusion PX, SH3-N, SH3-C, PP Protein (GST-PX, GST-SH3-N, GST-SH3-C, GST-PP)

(1) Cloning of Recombinant DNA

Human p47phox cDNA (Germany RZPD Co., Ltd.) (SEQ ID NO: 3) was used as a template, and oligonucleotides corresponding to the respective forward and reverse primers were synthesized as described below, followed by polymerase chain reaction (PCR) to obtain amino acids of p47phox ( PX encoding SEQ ID NO: 1) (amino acid 1-125 site coding of p47phox) (SEQ ID NO: 5), SH3-N (amino acid 156-215 site coding of p47phox) (SEQ ID NO: 4), SH3-C (amino acid of p47phox) CDNAs corresponding to 226-285 site coding) (SEQ ID NO: 6) and PP (amino acid 292-390 site coding of p47phox) (SEQ ID NO: 7) regions were cloned. These were cloned into expression vectors (pGEX4T-2) with T4 DNA ligase, respectively.

Primer for PX cDNA

F: 5'-AAACCGGAATTCGGGGACACCTTCATC-3 '(SEQ ID NO: 8)

R: 5'-AAACCGGAATTCTCACAAGTATGTCTCTGG-3 '(SEQ ID NO: 9)

Primer for cDNA of SH3-N (KIRAMS-NP47-SH3N)

F: 5'-AAACCGGAATTCCCCAAAGATGGCAAG-3 '(SEQ ID NO: 10)

R: 5'-AAACCGGAATTCTCATTCCGTCTCGTCAGG-3 '(SEQ ID NO: 11)

Primer for cDNA of SH3-C

F: 5'-AAACCGGAATTCGACCCTGAGCCCAAC-3 '(SEQ ID NO: 12)

R: 5'-AAACCGGAATTCTCAGGCCTGGGACACGTC-3 '(SEQ ID NO: 13)

Primer for PP cDNA

F: 5'-AAACCGGAATTCGGGGACACCTTCATC-3 '(SEQ ID NO: 14)

R: 5'-AAACCGGAATTCTCACAAGTATGTCTCTGG-3 '(SEQ ID NO: 15)

1 μl of this recombinant DNA was mixed with 100 μl of C-cell (Bl 21) and allowed to react on ice for 30 minutes. Heat shock was applied for 90 seconds in a 42 ° C. water bath, and then stored on ice for 5 minutes. 3 μl of this was put in 3 ml of LB medium and incubated at 37 ° C. for 1 hour in an incubator. 100 μl of the cultured LB medium was transformed on the LB plate to which ampicillin was added and cultured for 16 hours. DNA was extracted from the clones generated on the LB plate to confirm that the cDNA of PX, SH3-N, SH3-C and PP was normally cloned.

(2) Purification and Identification of Glutathione-S-Transferase (GST) Fusion PX, SH3-N, SH3-C, PP

The cDNAs of PX, SH3-N, SH3-C and PP cloned in Example 1 (1) were cloned into a pGEX vector containing a GST sequence (FIG. 1A) and transformed into BL21 (DE3), an E. coli strain. The conversion was made. Then, incubated with isopropyl-1-thio-β-D-galactopyranoside (IPTG) at 37 ° C. for 4 hours to induce protein expression, and glutathione- Proteins were isolated with sepharose beads (glutathione sepharose bead). Sample buffer was added in equal amounts to the isolated protein and boiled at 100 ° C. for 3 minutes and then subjected to Western blotting to induce GST fusion PX, SH3-N, SH3-C, PP protein (GST -PX, GST-SH3-N, GST-SH3-C, GST-PP) were confirmed (FIG. 1B).

Example  2: SH3 -N ( KIRAMS - NP47 - SH3N )of PKCd  Confirmation of active inhibitory effect

(1) GST-pull down assay

The lysate of the lysed cells was reacted with GST fusion protein connected to glutathione-sepharose beads for 2 hours at 4 ° C, washed 5 times with lysis buffer, and then lysed with 1X SDS sample buffer. After electrophoresis by addition, western blotting was performed.

(2) kinase assay down

1 μg of purified GST-PX, GST-SH3-N, GST-SH3-C and GST-PP, 1 μg of activated PKC α, β, δ proteins, 1 μg of myelin basic protein (MBP), a substrate of PKC 20 ml of kinase reaction buffer (50 mM HEPES (pH 7.5), 10 mM MgCl ₂ , 1 mM dithiothreitol, 2.5 mM EGTA, 1 mM NaF, 0.1) containing gamma ³² P ATP mM Na ₃ VO ₄ And 10mM b-glycerophosphate) and reacted at 30 ° C. for 30 minutes, and then 20 μl of 2X SDS sample buffer was added to terminate the reaction. Thereafter, the mixture was boiled at 90 ° C. for 5 minutes, followed by electrophoresis and autoradiograpy.

(3) Inhibitory Effect of SH3-N (KIRAMS-NP47-SH3N) on PKCd Activity

The kinase assay of Example 4 was performed by mixing purified GST-fusion protein (GST-PX, GST-SH3-N, GST-SH3-C, GST-PP), activated PKC α, β, δ protein, and MBP. As a result, when GST-PX, GST-SH3-C and GST-PP were mixed with PKC / MBP, a large amount of PKC and MBP phosphorylated to [g- ³² P] ATP, whereas GST-SH3- When N was mixed with PKC / MBP, it was confirmed that PKC and MBP hardly phosphorylated with [g- ³² P] ATP. That is, GST-PX protein, GST-SH3-C protein and GST-PP protein do not affect PKCd activity, whereas GST-SH3-N protein phosphorylates MBP, a substrate of PKCd, as well as its own activity. It was confirmed that it can be inhibited (Fig. 2A). In addition, when treated with 0.5, 1, 2mg of GST-SH3-N protein, it was confirmed that the PKCd itself and the phosphorylation of MBP may be inhibited depending on the concentration (Fig. 2B).

In addition, PKCα, PKCβ, and PKCζ, which are isozymes of PKCδ, were mixed with GST-SH3-N, and the kinase assay of Example 4 was performed. It was confirmed that the activity of PKCβ, PKCζ and the phosphorylation of MBP, which is their substrate (Fig. 3).

Example  3: K- Ras On by PKC  Check the activity boost effect

(1) Cell culture method

Rat2 fibroblat cells transfected with rat2 fibroblast cells and oncogenic K-Ras were cultured using RPMI 1640 medium containing 5% FBS and penicillin streptomycin.

(2) Plasmid Preparation and Transfection Methods

Recombinant DNA was prepared by cloning the K-Ras and PKC genes and the selection marker puromycin N-acetyl-transferase gene in the MFG retroviral vector, and each of them was MFG-K-Ras (V12)-. puro and MFG-DN-PKCd were named puro. MFG-puro cloned with only the puro gene without K-Ras and PKC genes was used as a control.

These retroviral vectors were transfected into a 293pgp retrovirus packaging cell line using Lipofectamine 2000 (Invitrogen, Carlsbad, Calif.) And harvested after 72 hours. 1 ml of this virus and 8 mg / ml of polybrene (Sigma Chemical Co., St. Louis, Mo.) were added to transduce Rat2 fibroblast and overexpress K-Ras and DN-PKCd.

In addition, Flag-SH3-N vector was prepared by inserting the cDNA of SH3-N cloned in Example 1 (1) into the pFLAGN1 vector having the tag as a tag, and using this lipofectamine 2000, Rat2 fibroblast Transfection of cells induced overexpression of SH3-N in cells.

(3) Western blot analysis

 In Example 3 (2), a recombinant DNA-transduced Rat2 fibroblast was added to a lysis buffer (40 mM Tris-Cl (pH 8.0), 120 mM NaCl, 0.1% Nonidet-P4) to which an inhibitor of protease was added. After dissolving, the mixture was centrifuged to obtain a supernatant containing protein. The separated proteins were separated by SDS-electrophoresis and attached to nitrocellulose membrane. The protein-attached membrane was blocked with a 5% fat free milk solution and then reacted with the primary antibody for 1 hour at room temperature. After washing this three times, the secondary antibody was reacted for 1 hour to confirm the expression level of proteins using the ECL method.

(4) Soft agar colony formation

1.8% and 0.9% of Noble agar solutions were prepared. A bottom agar was prepared by mixing 1.8% of the noble agar solution and 2X RPMI (10% FBS) in a 1: 1 ratio. 2 ml of this mixture was evenly added to a 60 mm dish and hardened for about 10 minutes. . Mix 0.9% noble agar solution with 2X RPMI (10% FBS) in a 1: 1 ratio, add Rat2 fibroblast at 10 ⁴ cells / dish, and evenly put 2ml on the bottom agar of the dish. Thereafter, the cells were cultured in a 37 ° C., 5% CO ₂ incubator, and the number of colonies whose colony formation was 0.3 mm or larger was measured.

(5) Immunoprecipitation and Immune Complex Kinase Assay

300 mg of the protein obtained as a result of the cell lysis was reacted with the primary antibodies (anti-PKC α, anti-PKC β, anti-PKC δ) at 4 ° C. for 2 hours, followed by protein A agarose beads. Immunoprecipitate by addition. Three times immunoprecipitated protein with lysis buffer, kinese reaction buffer (50 mM HEPES (pH 7.5), 10 mM MgCl ₂ , 1 mM dithiothreitol, 2.5 mM EGTA, 1 mM NaF, 0.1 mM Na ₃ VO ₄ , and 10 mM b-glycerophosphate) After washing twice with 20 μl of kinase reaction buffer containing 1 μg of MBP and 2 μCi of [g- ³² P] ATP, the reaction was carried out at 30 ° C. for 30 minutes, and then 20 μl of 2X SDS sample buffer was added. Was terminated. Then, after boiling for 5 minutes at 90 ℃ electrophoresis and radiographic methods were performed.

(6) effect of increasing PKC activity by K-Ras

As in Example 3 (2), the rat2 fibroblast was transduced with a virus containing MFG-K-Ras (V12) -puro to overexpress K-Ras, a cell carcinogenic gene, and then subjected to Immune Complex Kinase Assay. PKCδ It was confirmed that the activity of the increased (Fig. 4A and B). When the colony forming assay was performed by mixing DN-PKCδ, which is a PKCδ inhibitory gene, with Rat2 fibroblast overexpressing K-Ras or adding DN-PKCδ gene. It was confirmed that the decrease (Fig. 4C).

Experimental Example  1: in vitro SH3 Of -N PKC  Active inhibitory effect

As in Example 3 (2), Flag-SH3-N was transfected into Rat2 fibroblast cells, and Western blot using Flag antibody confirmed that SH3-N was normally expressed. In addition, when SH3-N is normally expressed, it was observed that phosphorylation of PKCδ and its substrate MBP is greatly suppressed (FIG. 5A). When MFG-K-Ras (V12) -puro was transfected into Rat2 fibroblast cells and overexpressed K-Ras intracellularly, about 140 cell colonies were produced, but MFG-K-Ras (V12) -puro and Flag Transfection of -SH3-N simultaneously reduced the number of colonies to about 20 (FIGS. 5B and C).

Experimental Example  2: in vivo in SH3 Of -N PKC  Active inhibitory effect

(1) In vivo test method

Tumor volume was measured at 3 days intervals after transplanting the rat2 fibroblasts overexpressing K-Ras and the rat2 fibroblasts overexpressing Flag-SH3-N and K-Ras in the femur of nude mice.

(2) Inhibitory Effect of SH3-N on PKC Activity in Vivo

Rat2 fibroblast cells overexpressed K-Ras were transplanted into nude mice, and it was confirmed that tumors were generated from 9 days after transplantation. Nude mice transplanted with cells overexpressing K-Ras and SH3-N simultaneously showed slower tumor growth compared to the case where only K-Ras was overexpressed at 9 days after transplantation (FIG. 6). ).

FIG. 1A shows the structure of the GST-fusion protein of the present invention, and FIG. 1B shows Western blot results for the GST fusion PX, SH3-N, SH3-C, PP proteins. Where * denotes a GST fusion protein.

FIG. 2A is a result of Western blot when GST-fusion protein (GST-PX, GST-SH3-N, GST-SH3-C, GST-PP) is mixed with PKC and MBP, and FIG. 2B is PKC, MBP and GST Western blot result of mixing -SH3-N by concentration.

3 is a Western blot result when GST-SH3-N is added to PKCα, PKCβ, PKCδ, PKCζ and MBP.

4A is a Western blot result showing PKC activated by overexpression of K-Ras, and FIG. 4B is an Immune Complex Kinase Assay result showing the effect of reducing K-Ras expression by DN-PKCδ. In addition, Figure 4C is a colony forming assay results showing the effect of reducing K-Ras expression by DN-PKCδ.

5A is a result of Western blot using Flag antibody after transfecting Flag-SH3-N into Rat2 fibroblast cells. 5B and C are also results of colony forming assays showing the activity of PKC of SH3-N. Here, PCDNA means pFLAGN1 itself which does not insert cDNA of SH3-N.

Figure 6 shows the change in tumor size in nude mice receiving K-Ras overexpressed Rat2 fibroblast cells.

<110> Korea Institute of Radiological and Medical Sciences <120> Protein for inhibiting PKC activity and pharmaceutical          composition including the same <130> PC07-0253 <160> 15 <170> KopatentIn 1.71 <210> 1 <211> 390 <212> PRT <213> Homo sapiens <400> 1 Met Gly Asp Thr Phe Ile Arg His Ile Ala Leu Leu Gly Phe Glu Lys   1 5 10 15 Arg Phe Val Pro Ser Gln His Tyr Val Tyr Met Phe Leu Val Lys Trp              20 25 30 Gln Asp Leu Ser Glu Lys Val Val Tyr Arg Arg Phe Thr Glu Ile Tyr          35 40 45 Glu Phe His Lys Thr Leu Lys Glu Met Phe Pro Ile Glu Ala Gly Ala      50 55 60 Ile Asn Pro Glu Asn Arg Ile Ile Pro His Leu Pro Ala Pro Lys Trp  65 70 75 80 Phe Asp Gly Gln Arg Ala Ala Glu Asn Arg Gln Gly Thr Leu Thr Glu                  85 90 95 Tyr Cys Ser Thr Leu Met Ser Leu Pro Thr Lys Ile Ser Arg Cys Pro             100 105 110 His Leu Leu Asp Phe Phe Lys Val Arg Pro Asp Asp Leu Lys Leu Pro         115 120 125 Thr Asp Asn Gln Thr Lys Lys Pro Glu Thr Tyr Leu Met Pro Lys Asp     130 135 140 Gly Lys Ser Thr Ala Thr Asp Ile Thr Gly Pro Ile Ile Leu Gln Thr 145 150 155 160 Tyr Arg Ala Ile Ala Asp Tyr Glu Lys Thr Ser Gly Ser Glu Met Ala                 165 170 175 Leu Ser Thr Gly Asp Val Val Glu Val Val Glu Lys Ser Glu Ser Gly             180 185 190 Trp Trp Phe Cys Gln Met Lys Ala Lys Arg Gly Trp Ile Pro Ala Ser         195 200 205 Phe Leu Glu Pro Leu Asp Ser Pro Asp Glu Thr Glu Asp Pro Glu Pro     210 215 220 Asn Tyr Ala Gly Glu Pro Tyr Val Ala Ile Lys Ala Tyr Thr Ala Val 225 230 235 240 Glu Gly Asp Glu Val Ser Leu Leu Glu Gly Glu Ala Val Glu Val Ile                 245 250 255 His Lys Leu Leu Asp Gly Trp Trp Val Ile Arg Lys Asp Asp Val Thr             260 265 270 Gly Tyr Phe Pro Ser Met Tyr Leu Gln Lys Ser Gly Gln Asp Val Ser         275 280 285 Gln Ala Gln Arg Gln Ile Lys Arg Gly Ala Pro Pro Arg Arg Ser Ser     290 295 300 Ile Arg Asn Ala His Ser Ile His Gln Arg Ser Arg Lys Arg Leu Ser 305 310 315 320 Gln Asp Ala Tyr Arg Arg Asn Ser Val Arg Phe Leu Gln Gln Arg Arg                 325 330 335 Arg Gln Ala Arg Pro Gly Pro Gln Ser Pro Gly Ser Pro Leu Glu Glu             340 345 350 Glu Arg Gln Thr Gln Arg Ser Lys Pro Gln Pro Ala Val Pro Pro Arg         355 360 365 Pro Ser Ala Asp Leu Ile Leu Asn Arg Cys Ser Glu Ser Thr Lys Arg     370 375 380 Lys Leu Ala Ser Ala Val 385 390 <210> 2 <211> 60 <212> PRT <213> Homo sapiens <400> 2 Ile Ile Leu Gln Thr Tyr Arg Ala Ile Ala Asp Tyr Glu Lys Thr Ser   1 5 10 15 Gly Ser Glu Met Ala Leu Ser Thr Gly Asp Val Val Glu Val Val Glu              20 25 30 Lys Ser Glu Ser Gly Trp Trp Phe Cys Gln Met Lys Ala Lys Arg Gly          35 40 45 Trp Ile Pro Ala Ser Phe Leu Glu Pro Leu Asp Ser      50 55 60 <210> 3 <211> 1340 <212> DNA <213> Homo sapiens <400> 3 ccacccagtc atgggggaca ccttcatccg tcacatcgcc ctgctgggct ttgagaagcg 60 cttcgtaccc agccagcact atgtgtacat gttcctggtg aaatggcagg acctgtcgga 120 gaaggtggtc taccggcgct tcaccgagat ctacgagttc cataaaacct taaaagaaat 180 gttccctatt gaggcagggg cgatcaatcc agagaacagg atcatccccc acctcccagc 240 tcccaagtgg tttgacgggc agcgggccgc cgagaaccgc cagggcacac ttaccgagta 300 ctgcagcacg ctcatgagcc tgcccaccaa gatctcccgc tgtccccacc tcctcgactt 360 cttcaaggtg cgccctgatg acctcaagct ccccacggac aaccagacaa aaaagccaga 420 gacatacttg atgcccaaag atggcaagag taccgcgaca gacatcaccg gccccatcat 480 cctgcagacg taccgcgcca ttgccgacta cgagaagacc tcgggctccg agatggctct 540 gtccacgggg gacgtggtgg aggtcgtgga gaagagcgag agcggttggt ggttctgtca 600 gatgaaagca aagcgaggct ggatcccagc atccttcctc gagcccctgg acagtcctga 660 cgagacggaa gaccctgagc ccaactatgc aggtgagcca tacgtcgcca tcaaggccta 720 cactgctgtg gagggggacg aggtgtccct gctcgagggt gaagctgttg aggtcattca 780 caagctcctg gacggctggt gggtcatcag gaaagacgac gtcacaggct actttccgtc 840 catgtacctg caaaagtcgg ggcaagacgt gtcccaggcc caacgccaga tcaagcgggg 900 ggcgccgccc cgcaggtcgt ccatccgcaa cgcgcacagc atccatcagc ggtcgcggaa 960 gcgcctcagc caggacgcct atcgccgcaa cagcgtccgt tttctgcagc agcgacgccg 1020 ccaggcgcgg ccgggaccgc agagccccgg gagcccgctc gaggaggagc ggcagacgca 1080 gcgctctaaa ccgcagccgg cggtgccccc gcggccgagc gccgacctca tcctgaaccg 1140 ctgcagcgag agcaccaagc ggaagctggc gtctgccgtc tgaggctgga gcgcagtccc 1200 cagctagcgt ctcggccctt gccgccccgt gcctgtacat acgtgttcta tagagcctgg 1260 cgtctggacg ccgagggcag ccccgacccc tgtccagcgc ggctcccgcc accctcaata 1320 aatgttgctt ggagtggaag 1340 <210> 4 <211> 183 <212> DNA <213> Homo sapiens <400> 4 atcatcctgc agacgtaccg cgccattgcc gactacgaga agacctcggg ctccgagatg 60 gctctgtcca cgggggacgt ggtggaggtc gtggagaaga gcgagagcgg ttggtggttc 120 tgtcagatga aagcaaagcg aggctggatc ccagcatcct tcctcgagcc cctggacagt 180 cct 183 <210> 5 <211> 375 <212> DNA <213> Homo sapiens <400> 5 atgggggaca ccttcatccg tcacatcgcc ctgctgggct ttgagaagcg cttcgtaccc 60 agccagcact atgtgtacat gttcctggtg aaatggcagg acctgtcgga gaaggtggtc 120 taccggcgct tcaccgagat ctacgagttc cataaaacct taaaagaaat gttccctatt 180 gaggcagggg cgatcaatcc agagaacagg atcatccccc acctcccagc tcccaagtgg 240 tttgacgggc agcgggccgc cgagaaccgc cagggcacac ttaccgagta ctgcagcacg 300 ctcatgagcc tgcccaccaa gatctcccgc tgtccccacc tcctcgactt cttcaaggtg 360 cgccctgatg acctc 375 <210> 6 <211> 183 <212> DNA <213> Homo sapiens <400> 6 tatgcaggtg agccatacgt cgccatcaag gcctacactg ctgtggaggg ggacgaggtg 60 tccctgctcg agggtgaagc tgttgaggtc attcacaagc tcctggacgg ctggtgggtc 120 atcaggaaag acgacgtcac aggctacttt ccgtccatgt acctgcaaaa gtcggggcaa 180 gac 183 <210> 7 <211> 297 <212> DNA <213> Homo sapiens <400> 7 cgccagatca agcggggggc gccgccccgc aggtcgtcca tccgcaacgc gcacagcatc 60 catcagcggt cgcggaagcg cctcagccag gacgcctatc gccgcaacag cgtccgtttt 120 ctgcagcagc gacgccgcca ggcgcggccg ggaccgcaga gccccgggag cccgctcgag 180 gaggagcggc agacgcagcg ctctaaaccg cagccggcgg tgcccccgcg gccgagcgcc 240 gacctcatcc tgaaccgctg cagcgagagc accaagcgga agctggcgtc tgccgtc 297 <210> 8 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> PX primer forward <400> 8 aaaccggaat tcggggacac cttcatc 27 <210> 9 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> PX primer reverse <400> 9 aaaccggaat tctcacaagt atgtctctgg 30 <210> 10 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> SH3-N (KIRAMS-NP47-SH3N) primer forward <400> 10 aaaccggaat tccccaaaga tggcaag 27 <210> 11 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> SH3-N (KIRAMS-NP47-SH3N) primer reverse <400> 11 aaaccggaat tctcattccg tctcgtcagg 30 <210> 12 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> SH3-C primer forward <400> 12 aaaccggaat tcgaccctga gcccaac 27 <210> 13 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> SH3-C primer reverese <400> 13 aaaccggaat tctcaggcct gggacacgtc 30 <210> 14 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> PP primer forward <400> 14 aaaccggaat tcggggacac cttcatc 27 <210> 15 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> PP primer reverse <400> 15 aaaccggaat tctcacaagt atgtctctgg 30  

Claims (9)

A nucleic acid molecule encoding a protein capable of inhibiting PKC activity consisting of the amino acid sequence of SEQ ID NO: 2. The nucleic acid molecule of claim 1, wherein the nucleic acid molecule encoding the amino acid sequence of SEQ ID NO: 2 is a DNA sequence. The nucleic acid molecule of claim 2 which is cDNA. A nucleic acid molecule encoding a protein capable of inhibiting PKC activity consisting of the nucleotide sequence of SEQ ID NO: 4. A recombinant vector comprising the nucleic acid molecule of claim 4. A transformant transformed by the expression vector of claim 5. A protein capable of inhibiting PKC activity encoded by the nucleic acid molecule according to any one of claims 1 to 4. 8. The protein according to claim 7, which can inhibit PKC activity consisting of the amino acid sequence of SEQ ID NO: 2. A pharmaceutical composition for preventing or treating tumors, comprising a protein capable of inhibiting PKC activity encoded by the nucleic acid molecule of any one of claims 1 to 4 and a pharmaceutically acceptable carrier.

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