KR20100085317A - Method for screening inhibitor of ampk activity - Google Patents

Abstract

PURPOSE: A method for screening an inhibitor of AMPK activation is provided to select a material which suppresses inter-combination of AMPKγ1 and DNA-PKcs and to use as an anticancer drug. CONSTITUTION: A method for screening drug for suppressing cancer proliferation and/or metastasis or protecting nerve comprises: a step of treating a candidate material which suppresses inter-combination of AMPKγ1 and DNA-PKcs (experimental group); a step of measuring activation of inter-combination of AMPKγ1 and DNA-PKcs(positive control group) and inter-combination of the experimental group; and a step of selecting candidate material over the positive control group.

Description

Method for screening inhibitor of AMPK activity

The present invention relates to a method for screening an AMPK activity inhibitor.

DNA-dependent protein kinase (DNA-PK) is a nuclear serine / threonine protein kinase activated by DNA double strand break (DSB) (Hartley KO et al., Cell 82: 849-856, 1995; Jackson SP Int J Biochem Cell Biol 29: 935-938, 1997; Jeggo PA Adv Genet 38: 185-218, 1998). DNA-PK is initially recognized and activated as a DNA damage sensor by recognizing DNA damage induced by ionizing and UV radiation (Burma S & Chen DJ, DNA Repair , Amst 3: 909-918, 2004). DNA-PK plays an important role in the DNA repair process known as nonhomologous DNA end-joining (NHEJ) by binding to damaged DNA and then targeting other repair activity to the site of injury. DNA-PK consists of a regulatory component of a 470-kDa catalytic subunit (DNA-PKcs; DNA-PK catalytic subunit) and a Ku70 / Ku80 heterodimer (Smith GC & Jackson SP, Genes Dev 13: 916-934, 1999; Lees-Miller SP & Meek K, Biochimie 85: 1161-1173, 2003). The DNA-PKcs belong to the family of phosphatidyl-inositol 3-OH kinase-related kinases (PIKK), including ataxia telangiectasia mutated (ATM) and ATM-RAD-3-related (ATR) -kinases, etc. in mammals (Smith GC & Jackson SP, Genes Dev 13: 916-934, 1999; Bentley NJ et al., Embo J 15: 6641-6651, 1996; Cliby WA et al., Embo J 17: 159-169, 1998), the family The members of are high molecular weight polypeptides that share a conserved C-terminal region, homologous to the catalytic activity domain of phosphatidylinositol-3-OH kinase (PIK). The ATM, ATR and DNA-PKcs function to phosphorylate targets such as H2AX, Chk1, Chk2, Nbs1 and MDC1 in relation to cellular responses to DSB (Abraham RT, DNA Repair, Amst 3: 883-887, 2004). DNA-PK bound to p53 plays a role in detecting abnormal DNA structures (Achanta G et al., Cancer Res 61: 8723-8729, 2001), and p53 is an effector for DNA-PKcs-mediated signaling. DNA-PKcs selectively modulates p53-independent apoptosis (Wang S et al., Proc Natl Acad Sci USA 97: 1584-1588, 2000).

AMP-activated protein kinase (AMPK) is a serine / threonine protein kinase that acts as an energy sensor in eukaryotic cells (Warden SM et al., Biochem J 354: 275-283, 2001). AMPK is a heterodimer consisting of an α subunit with catalytic activity and two regulatory and targeting subunits β and γ. Mammalian AMPK includes two α subunits, three γ subunits and two β subunits (α1, α2, γ1, γ2, γ3, β1 and β2) (Mitchelhill KI et al., J Biol Chem) 272: 24475-24479, 1997). The α subunits each comprise a core 1-312 having catalytic activity at the N-terminus, and at the C-terminus are involved in autoregulation and bind to β and γ subunits 313 -548) (Crute BE et al., J Biol Chem 273: 35347-35354, 1998). The γ subunits each comprise four tandem cystathione β-synthase (CBS) domains that bind together with two AMP molecules and allow a kinase complex that recognizes the amount of adenine nucleotides. The β subunit is expressed in a cell membrane-like cell (Polekhina G et al., Curr Biol 13: 867-871, 2003; Long YC & Zierath JR, J Clin ) through its N-terminal myristoylation . Invest 116: 1776-1783, 2006) and intracellular glycogen via glycogen binding domains (Alessi DR et al., Annu Rev Biochem 75: 137-163, 2006), as well as α and γ subunits. It has been reported to act as a framework for. Binding of the AMP to the CBS domain of the AMPKγ subunit induces activation of the AMPK complex, neutralized by the active high concentration of ATP, and enables a system for monitoring changes in the intracellular AMP: ATP ratio. DG et al., Biochem J 338, Pt 3): 717-722, 1999; Scott JW et al., J Clin Invest 113: 274-284, 2004). AMPK activation by increasing intracellular AMP concentrations promotes catabolic pathways that inhibit anabolic pathways and restore intracellular energy levels. Activation of AMPK induces phosphorylation of numerous proteins, resulting in inhibition of fatty acid synthesis, cholesterol synthesis and glucose production, as well as glucose uptake, glycolysis and fatty acid oxidation (Hardie DG & Hawley SA, Bioessays 23: 1112 -1119, 2001; Kahn BB et al., Cell Metab 1: 15-25, 2005).

At least two kinases have been shown to phosphorylate AMPK: LKB1, a tumor suppressor kinase, complex of two accessory subunits STRAD and MO25 (Hawley SA et al., J Biol 2:28, 2003; Hong SP et al. Proc Natl Acad Sci USA 100: 8839-8843, 2003; Shaw RJ et al., Proc Natl Acad Sci USA 101: 3329-3335, 2004; Woods A et al., Curr Biol 13: 2004-2008, 2003); CaMKKβ (Ca ²⁺ -calmodulin-dependent protein kinase β) (Hawley SA et al., Cell Metab 2: 9-19, 2005; Woods A et al., Cell Metab 2: 21-33, 2005). LKB1 and CaMKKβ directly phosphorylate Thr172 of α subunit having catalytic activity of AMPK. Recently, Tak1, a member of the mitogen-activated protein kinase kinase (MAPKK) family, has also been reported to be capable of AMPK phosphorylation and activation (Momcilovic M et al., J Biol Chem 281: 25336-25343, 2006). The LKB1 is structurally active but not stimulated by AMP (Woods A et al., Curr Biol 13: 2004-2008, 2003; Lizcano JM et al., Embo J 23: 833-843, 2004; Sakamoto K et al., Am J Physiol Endocrinol Metab 287: E310-E317, 2004). In vitro studies have suggested the binding of AMP and AMPK as a fundamental regulatory mechanism to stimulate the phosphorylation of AMPK by LKB1 (Hawley SA et al., J Biol 2:28, 2003). LKB1, a tumor suppressor gene, is a major AMPK kinase and Peutz-Jeghers syndrome is caused by mutations in LKB1 (Hawley SA et al., J Biol 2:28, 2003; Shaw RJ et al., Proc Natl Acad Sci USA 101: 3329-3335, 2004; Woods A et al., Curr Biol 13: 2004-2008, 2003). AMPK can also be activated in an AMP-independent manner by hyperosmotic stress or the antidiabetic drug metformin (Fryer LG et al., J Biol Chem 277: 25226-25232, 2002). In some tissues CaMMKβ is an alternative upstream kinase that activates AMPK in Ca ²⁺ -dependent conditions (24. Hawley SA et al., Cell Metab 2: 9-19, 2005; Woods A et al., Cell Metab 2: 21-33, 2005). The action of CAMKKβ on top of AMPK suggests that it can act as a secondary pathway that activates AMPK with a change in AMP: ATP ratio by an increase in intracellular Ca ²⁺ .

Glucose consumption, a major cellular energy source, is indicated by increased AMP levels and decreased ATP levels and can result from a variety of stresses such as nutrient deficiency, hypoxia and oxidative stress (Long YC & Zierath JR, J Clin Invest 116: 1776-1783, 2006). AMPK, a protein kinase activated by AMP increase by glucose depletion, is an intracellular energy sensor that plays an important role in intracellular energy homeostasis (Kahn BB et al., Cell Metab 1: 15-25, 2005) . It is well known that AMPK is activated by binding AMP to AMPKγ by glucose consumption. AMP-mediated allosteric activation, enhanced phosphorylation by upstream kinases, and inhibition of Thr172 dephosphorylation by protein phosphatase [Hardie DG et al., Biochem J 338, Pt 3 : 717-722, 1999], AMP binds to a regulatory γ subunit of AMPK that induces phosphorylation of AMPKα that occurs after cellular energy is consumed (Adams J et al ., Protein Sci 13: 155-165, 2004).

The AMPK is widely expressed in human organs and tissues, such as heart, skeletal muscle, liver, and fat, thereby exhibiting this action. Increasing AMPK activity in the heart increases glucose uptake into the cell, activating glycolysis and promoting fatty acid oxidation, and in skeletal muscle, increases glucose uptake and increases ATP production due to glycolysis and fat oxidation, as well as the development of mitochondria. It also promotes energy consumption by increasing the expression of UCP (Uncoupling Protein). Hepatic AMPK is known to inhibit fatty acid and cholesterol synthesis, inhibit your production, and in fat, inhibit fatty acid synthesis and promote lipolysis. Accordingly, a patent (Korean Patent No. 10-0681439) has been reported regarding the 'agent having AMPK activation and having trans-resveratrol as an active ingredient having anti-obesity and anti-diabetic effect.'

In addition, by inhibiting the activity of the AMPK to enhance the pharmacological effect by the anticancer agent, to inhibit the proliferation and / or metastasis of cancer (Korea Patent Publication No. 10-2006-0066610) and nerves by pharmacological inhibition of AMPK The protection method (Korean Patent Publication No. 10-2007-0085093) and the like have been reported.

Accordingly, the present inventors discovered AMPKγ1 while screening a protein binding to DNA-PKcs by the yeast Two-Hybrid method, and AMPKγ1 is an endogenous interaction partner of DNA-PKcs in glioma cells rich in DNA-PKcs, It was confirmed that AMPK activity induced by glucose consumption is inhibited by inhibition of DNA-PKcs activity. Since the DNA-PKcs is an activator of AMPKγ1, the present invention was completed by confirming that the DNA-PKcs can be usefully used for screening a substance for controlling the interaction of DNA-PKcs and AMPKγ1.

An object of the present invention is to inhibit the mutual binding activity between AMP-activated protein kinase γ1 (AMPKγ1) and DNA-PK catalytic subunit (DNA-PKcs), thereby inhibiting the proliferation and / or metastasis of cancer or neuroprotection. It is to provide a method for screening drugs.

In order to achieve the above object, the present invention

1) treating AMPKγ1, its binding partner DNA-PKcs and candidates that inhibit the mutual binding between the AMPKγ1 and DNA-PKcs (experimental group);

2) measuring the mutual binding activity (positive control group) between AMPKγ1 and DNA-PKcs and the mutual binding activity of the experimental group of step 1), respectively; And,

3) It provides a method for screening a drug for inhibiting the proliferation and / or metastasis of cancer comprising the step of selecting a candidate to inhibit the cross-linking activity than a positive control group.

In addition, the present invention

1) treating AMPKγ1, its binding partner DNA-PKcs and candidates that inhibit the mutual binding between the AMPKγ1 and DNA-PKcs (experimental group);

2) measuring the mutual binding activity (positive control group) between AMPKγ1 and DNA-PKcs and the mutual binding activity of the experimental group of step 1), respectively; And,

3) provides a method of screening a neuroprotective drug comprising the step of selecting a candidate to inhibit the cross-linking activity than a positive control group.

Hereinafter, the present invention will be described in detail.

In a specific embodiment of the invention, the M059K and M059J cell lines were incubated for 12 hours in normal or glucose consuming medium, and then the cells were disrupted. Proteins were immunoprecipitated from the lysate using DNA-PK-specific antibodies, followed by immunoblotting using anti-AMPKγ1 antibodies. As a result, it was shown that endogenous DNA-PKcs binds to AMPKγ1 in M059K, but not in MO59J cells. In the glucose-depleted state, the amount of DNA-PKcs bound to AMPKγ1 was not significantly changed (see FIG. 1A). In the mutual co-immunoprecipitation test using an anti-AMPKγ1 antibody, endogenous DNA-PKcs were easily immunoprecipitated from cell lysate (see FIG. 1B). The binding was specific so that DNA-PKcs was not detected in the samples immunoprecipitated using rabbit IgG. From the above results, it was confirmed that AMPKγ1 is an endogenous interaction partner of DNA-PKcs.

Energy deficiency, defined as an increase in AMP / ATP ratio, can be caused by several factors such as glucose consumption, hypoxia and oxidative stress (Long YC & Zierath JR, J Clin Invest 116: 1776-1783, 2006 ). The cellular AMP / ATP ratio is one of the major determinants of AMPK activity, and AMPK phosphorylation is induced by glucose consumption. To determine whether binding of AMPKγ1 to AMPKγ1 of DNA-PKcs affects AMPKα phosphorylation and activation, we phosphorylated AMPK in situations where DNA-PKcs lacked and glucose was consumed in cells with sufficient DNA-PKcs. The ability to do this was measured. As a result, AMPK phosphorylation at the Thr172 position, induced by glucose depletion, was inhibited in human glioma cells lacking DNA-PKcs (MO59J) and was significantly greater in M059K cells with sufficient DNA-PKcs (see FIG. 2A). . Similarly, when two cell types were incubated with a uniform decrease in glucose concentration for 24 hours, the effect on AMPK was greater in M059K cells than MO59J cells in the absence of glucose (see FIG. 2B), but the overall AMPKα The amount of expression remained constant. In addition, AMPK activity is a long-chain fatty acid synthetase, a rate-limiting enzyme indicated by the degree of Ser79 phosphorylation of an acetyl-CoA carboxylase (ACC) substrate (Hardie DG et al., J Physiol 574: 7-15, 2006). In this regard, we also examined ACC phosphorylation as a marker of AMPK activity. As a result, the basic and promoted phosphorylation of ACC at Ser79 was higher in M059K cells than in M059J cells, indicating once again that DNA-PK is involved in AMPK activation (see FIG. 2). UV irradiation in M059K cells does not increase AMPK phosphorylation and AMPK activation at Thr172 (no data), although various downstream targets in which DNA-PKcs are primarily involved in DNA repair and respond to single or double strand cleavage Even though phosphorylation of proteins, DNA-PKcs activation associated with DNA damage does not affect AMPK activation.

To further investigate the role of DNA-PKcs involved in AMPK activity, phosphorylation was measured using SAMS peptide assay on whole immunoprecipitated AMPK. As a result, the basic AMPK activity of M059J and M059K cells was 995 ± 22 and 1020 ± 25 cpm, respectively, and the AMPK activity was increased to 1160 ± 26 and 1480 ± 36 cpm, respectively, in M059J and M059K cells exposed to glucose consumption. . That is, AMPK activity induced by glucose depletion was inhibited in cells lacking DNA-PK (see FIG. 3).

Phosphorylation of AMPK induced by glucose depletion was shown to be reduced by inhibition of DNA-PKcs in M059K cells. To investigate the role of DNA-PKcs in phosphorylation of AMPK under glucose depletion conditions, siRNA can be used to suppress the expression level of DNA-PKcs or to be a powerful and irreversible enzyme inhibitor, wortmannin at high concentrations (above 10 μM). Was used to inhibit the activity of DNA-PKcs. As a result, in M059K cells transformed with DNA-PKcs siRNA, it was confirmed that the decrease in the amount of DNA-PKcs expression significantly reduced the phosphorylation of AMPK induced by glucose consumption (see FIG. 4A). In addition, AMPK (Thr172) activity in the cells suppressed the expression of DNA-PKcs increased less than the control (see Figure 4b). In addition, it was confirmed that the AMPK phosphorylation stimulated by glucose consumption was less increased in the Waltmanin-treated cells than in the non-Waltmanin-treated cells (see FIG. 4C). The results indicate that DNA-PKcs increases AMPK activity induced by glucose consumption.

To determine if DNA-PKcs really increases AMPK activation, we found that the activity of AMPK induced by glucose depletion in human fibroblast WI38 and IMR90 cell lines was reduced by inhibition of DNA-PKcs expression (FIG. 5). The results indicate that DNA-PKcs is involved in AMPK activity induced by glucose depletion in primary human diploid fibroblasts.

AMPK activity induced by glucose depletion was reduced by inhibition of DNA-PKcs in HeLa cell lines deficient in LKB1. LKB1 is a major protein kinase involved in phosphorylation of AMPK under conditions such as hypoxia, oxidative stress and nutrient deficiency (Alessi DR et al., Annu Rev Biochem 75: 137-163, 2006). As the above results suggest that DNA-PKcs has been shown to contribute to the phosphorylation of AMPK, the present inventors have found that HeLa cell line that does not express LKB1 (Hurley RL et al., J Biol Chem 280: 29060-29066, 2005). The effect of DNA-PK on AMPK phosphorylation was also examined. In order to verify the theory that DNA-PKcs functions as a promoter of AMPK in HeLa cell lines, HeLa cell lines transfected with DNA-PKcs siRNA were cultured in glucose consumption conditions, and phosphorylation of AMPKα was measured by immunoblotting. . As a result, under glucose consuming conditions, phosphorylation of AMPK was significantly reduced in HeLa cells in which the expression of DNA-PKcs was suppressed compared to control cells (see FIG. 6A). In addition, the phosphorylation of AMPK was also significantly lower in treated cells than in untreated control cells, even by the DNA-PKcs inhibitor Waltmannin (see Figure 6b). The results strongly support the inclusion of DNA-PKcs in the phosphorylation of AMPK under glucose consuming conditions. However, the concentration of Waltmannin also inhibits almost all PI3 kinases, as well as other kinases such as PI4 kinase and myosin light chain kinase (MLCK). Thus, it cannot be excluded that one of the kinases involved in AMPK activation in response to glucose consumption is involved.

LKB1 is not detected in human cervical cancer cell HeLa cell lines (Hurley RL et al., J Biol Chem 280: 29060-29066, 2005). Thus, CaMKK acts as an AMPK kinase to regulate AMPK in HeLa cell lines. However, the CaMKK inhibitor STO-609 does not completely block the phosphorylation and activation of AMPK in response to mannitol and 2-dioxyglucose (Hurley RL et al ., J Biol Chem 280: 29060-29066, 2005), suggesting that other AMPK regulatory mechanisms may exist in these cells. In addition, the present inventors, after administering STO-609, an inhibitor of CAMKK known to be involved in phosphorylation of AMPK, and Waltmannin, an inhibitor of DNA-PKcs, compared the effect on AMPK phosphorylation by glucose consumption. Inhibition of AMPK activity through the inhibition of DNA-PKcs showed that the inhibitory effect of AMPK phosphorylation activity of Waltmanin, a DNA-PKcs inhibitor, and STO-609, which was previously known to strongly inhibit AMPK, was similar. 6c). Therefore, DNA-PKcs may play an important role in AMPK activation during glucose deficiency. The findings direct DNA-PK as a modulator candidate of AMPK in mammalian cells.

Therefore, the method of the present invention can be usefully used in a method for screening drugs that can be used for inhibiting the proliferation and / or metastasis of cancer or neuroprotection by inhibiting the mutual binding activity of AMPKγ1 and DNA-PKcs.

The present invention

1) treating AMPKγ1, its binding partner DNA-PKcs and candidates that inhibit the mutual binding between the AMPKγ1 and DNA-PKcs (experimental group);

2) measuring the mutual binding activity (positive control group) between AMPKγ1 and DNA-PKcs and the mutual binding activity of the experimental group of step 1), respectively; And,

3) It provides a method for screening a drug for inhibiting the proliferation and / or metastasis of cancer comprising the step of selecting a candidate to inhibit the cross-linking activity than a positive control group.

AMPKγ1 and DNA-PKcs of step 1) are characterized in that described by SEQ ID NO: 1 and SEQ ID NO: 2, respectively. The AMPKγ1 and DNA-PKcs may be purchased and used in an already isolated and purified form, or may be transformed into transformants transformed with respective expression vectors including polynucleotides encoding amino acid sequences of SEQ ID NO: 1 and SEQ ID NO: 2. It may be used in an intrinsic form or separated and purified from the transformant, but is not limited thereto. One of the AMPKγ1 and DNA-PKcs can be bound to a solid substrate to facilitate subsequent steps such as washing or separation of the complex. Solid substrates include synthetic resins, nitrocellulose, glass substrates, metal substrates, glass fibers, microspheres and microbeads. In addition, the synthetic resins include polyester, polyvinyl chloride, polystyrene, polypropylene, PVDF and nylon. In a specific embodiment of the present invention, in order to bind an antibody specifically binding to a protein to a solid substrate, the microspheres are suspended and then transferred to a microtube to remove the supernatant by centrifugation, and then resuspended. N-hydroxy-sulfosuccinimide and 1-ethyl-3- (3-dimethylaminopropyl) -carbodiimide hydrochloride (1-ethyl-3- (3-dimethylaminopropyl) -carbodiimide hydrochloride) was treated sequentially, and then the supernatant was removed by centrifugation, washed and stored.

Step 1) The candidates are preferably, but not limited to, natural compounds, synthetic compounds, RNA, siRNA, DNA, polypeptides, enzymes, proteins, ligands, antibodies, antigens, metabolites of bacteria or fungi.

The measurement of the cross-linking activity of step 2) is a method for measuring the cross-linking activity between proteins, and any method known in the art can be used, for example, a protein in which one of AMPKγ1 and DNA-PKcs is immobilized on a solid substrate. A method of measuring the detector after binding the remaining protein to which a fluorescent substance is attached to the chip as a detector, specifically bound to a solid substrate, the remaining protein and the remaining protein, and binding the antibody labeled with the detector Using a method for measuring the detector, a surface plasmon resonance (SPR) method for measuring the plasmon resonance change on the surface in real time without labeling the detector, or a surface plasmon resonance imaging (SPRI) method for imaging and confirming the SPR system. But is not limited thereto. In addition, a fluorescence method performed by detecting a fluorescence with a fluorescent substance attached thereto or a radiation method performed by detecting a radiation with a radioisotope attached thereto as a detector may be used, but is not limited thereto.

In addition, the present invention

1) treating AMPKγ1, its binding partner DNA-PKcs and candidates that inhibit the mutual binding between the AMPKγ1 and DNA-PKcs (experimental group);

2) measuring the mutual binding activity (positive control group) between AMPKγ1 and DNA-PKcs and the mutual binding activity of the experimental group of step 1), respectively; And,

3) provides a method of screening a neuroprotective drug comprising the step of selecting a candidate to inhibit the cross-linking activity than a positive control group.

Substances that inhibit the interaction of AMPKγ1 and DNA-PKcs screened by the method of the present invention can be used as an anticancer agent or a neuro hobo drug.

Hereinafter, the present invention will be described in detail by examples and production examples.

However, the following Examples and Preparation Examples are merely illustrative of the present invention, and the content of the present invention is not limited to the following Examples.

Example 1 Selection of Protein Binding to DNA-PKcs

In order to identify novel DNA-PKcs-interacting proteins, the inventors of the present invention, using a matchmaker GAL4 Two-Hybrid System 3 (Clontech, USA) for human adult prostate cDNA expression libraries, follow the method suggested by the manufacturer. Hybrid screen was performed.

1) Specifically, in order to obtain cDNA of human DNA-PKcs (nts SEQ ID NO: 6, aa SEQ ID NO: 1), total RNA was obtained from human GM00637 cells (ATCC, USA) using RNeasy mini kit (QIAGEN, USA). After extraction, 50 μl of distilled water was filled in 1 μg of total RNA and 1 μl of oligo-d (T) (Invitrogen, USA, 0.5 μg / μl), and then placed in AccuPower RT-premix (Bioneer, Korea) for RT-PCR ( reverse transcription polymerasechain reaction. 5 minutes at 70 ° C, 5 minutes at 4 ° C, 60 minutes at 42 ° C, 5 minutes at 94 ° C, and 5 minutes at 4 ° C to obtain cDNA. Primer of SEQ ID NO: 7 (sense: 5'-GAATTCAAGCGAGTCCGTGGGAAGGCC-3 ') and SEQ ID NO: 8 (antisense: 5'-GTCGACTTCGCTGCTTATGATGAAGGG-3') using the cDNA (3-5 μl) or distilled water (negative control) as a template. PCR was performed using the pairs and PCR Master Mix (Promega, USA). More specifically, denature for 5 minutes at 95 ℃, turn 35 revolutions at the conditions of 95 ℃ 45 seconds, 65 ℃ 45 seconds, 72 ℃ 45 seconds, and then stretched for 5 minutes at 72 ℃, then at 4 ℃ It was carried out in the order of cooling. After the completion of the reaction, the PCR product produced by linking the GAL4 DNA-binding domain (DNA-binding domain) with EcoR I and Sal I restriction enzymes (promega, USA) was used as a bait plasmid. the ADE2, HIS3, lacZ and MEL1 reporter containing structure, inexpensive Caro My process three Levy jiae (Saccharomyces cerevisiae ) strain AH109 (Clontech, USA). The transformants were cultivated at 30 ° C. for 3-5 days in SD solid selection media lacking Trp to select yeast strains transformed with bait plasmids.

2) The selected yeast strains were transformed with a pACT2 plasmid (Clontech, USA) designed to connect the GAL4 activating domain and the prostate cDNA library. The transformants were cultured in SD solid selection media lacking Leu, Trp and His, followed by formation of additional blue colonies to select strains expressing α-galactosidase, X-α-Gal It was cultured in SD medium containing.

3) After separating the plasmid DNA from the selected clones, the sequence was analyzed to identify the gene encoding the protein of interest. The nucleotide sequence (SEQ ID NO: 9) was analyzed by BLAST of NCBI, and found to be AMPKγ1.

4) The AMPKγ1 was cloned back into the pACT2 vector, and then transduced with E. coli DH5α. After confirming that the expression is correct, the strain was introduced into an AH109 yeast strain containing up to pACT2 bait plasmid containing AMPKγ1. The assay was repeated by culturing in selection medium, and AMPKγ1 was again confirmed by reanalyzing the nucleotide sequences of the clones forming blue colonies.

Example 2 Confirmation of DNA-PKcs and AMPKγ1 Binding in Glioma Cells

The M059K cell line rich in DNA-PKcs and the M059J cell line deficient in M059K DNA-PKcs were investigated for the DNA-PKcs to interact with AMPKγ1.

1) Human glioma M059K and M059J cell lines (American Type Culture Collection; ATCC, USA) were DMEM / F12 (Dulbecco's modified Eagle) with 10% FBS, penicillin (100 U / mL) and streptomycin (100 μg / mL) medium with Ham's F-12 nutrient mixture, 1: 1; Invitrogen, USA). Each of these cell lines was further incubated for 12 hours in normal or glucose consuming medium. All incubations were performed in an incubator with humidity controlled at 5% CO ₂ and 37 ° C.

2) the cell line was subjected to cytolysis solution (25 mM Tris, pH 7.2, 150 mM NaCl, 5 mM MgCl ₂ , 0.5% NP-40, 1 mM DTT, 5% glycerol, 1 mM phenylmethanesulfonyl fluoride, 2 μg / ml pepstatin, 2 μg / ml leupeptin and 2 μg / ml aprotinin) and incubated at 4 ° C. for one hour. After centrifugation at 13,000 × g for 30 minutes, cells were purified from the supernatant using anti-DNA-PK antibody or anti-AMPKγ1 antibody (Abcam, USA) and normal immunoglobulin (normal rabbit IgG; Santa Cruz Biotechnology, USA). My protein was immunoprecipitated. After binding to the bound protein G-Agarose beads (Amersham Biosciences, USA), the cells were washed with excess cytolysis solution and boiled in SDS sample buffer. Proteins were fractionated by SDS-PAGE method, followed by immunoblotting using anti-DNA-PKcs and anti-AMPKγ1 antibodies.

3) The isolated protein concentration was measured using a dye-binding microassay (Bio-Rad, USA). The isolated protein (20 μg) was mixed with Laemmli sample buffer 1: 4 and boiled at 100 ° C. for 5 minutes. Boiled samples were loaded into wells of a 10% SDS-PAGE gel and electrophoresed at 125 V for 2 hours to separate them according to molecular weight. (PALL Life Sciences, USA). The membrane to which the protein was transferred was treated with Tris-buffered saline (10 mM Tris-HCl, pH 7.6, 150 mM NaCl) containing 0.1% Tween-20 and 5% skim milk, to which the protein was not bound. After blocking, bound primary antibody (anti-DNA-PK antibody or anti-AMPKγ1 antibody and α-tubulin antibody (Santa Cruz Biotechnology, Inc., USA)) diluted according to the manufacturer's protocol to the blocking solution. And then washed. Then, after incubation with a secondary antibody (anti-rabbit-IgG-HRP; Amersham Bioscience, UK) diluted 1: 4,000 in blocking solution, and washed again. The luminescence reaction was induced with an ECL detection kit (iNtRON Biotech, Korea) and the binding reaction was observed by exposure to a film (Las-4000, Fuji).

As a result, as shown in FIG. 1A, endogenous DNA-PKcs binds to AMPKγ1 in M059K, but not in MO59J cells. In the glucose-depleted state, the amount of DNA-PKcs bound to AMPKγ1 was not significantly changed.

In addition, in the mutual co-immunoprecipitation test using an anti-AMPKγ1 antibody as shown in FIG. The binding was specific so that DNA-PKcs was not detected in the samples immunoprecipitated using rabbit IgG.

Example 3 Confirmation of Phosphorylation of AMPKγ1 in Cells Lacking DNA-PKcs

M059K cell lines rich in DNA-PKcs and M059J cell lines deficient in M059K DNA-PKcs were examined for AMPK phosphorylation induced by glucose depletion.

<3-1> AMPK Phosphorylation by Glucose Consumption

M059K and M059J human glioma cells cultured according to the method of Example 2 in glucose depleted medium for 0, 6, 12 and 24 hours were washed with phosphate buffered saline, followed by cytolysis solution (20 mM HEPES, pH 7.4, 2 mM EGTA, 50 mM glycerol phosphate, 1% Triton X-100, 10% glycerol, 1 mM DTT, 1 mM phenylmethanesulfonyl fluoride, 10 μg / ml leupeptin, 10 μg / ml aprotinin, 1 mM Na3VO4 and 5 mM NaF) Incubated at 0 ° C. for 30 minutes. After centrifugation at 14,000 × g for 30 minutes, the supernatant was extracted from anti-phosphate-AMPKα (Cell Signaling Technology, USA), anti-AMPKα (Cell Signaling Technology, USA), anti-phosphate-ACC (acetyl-CoA carboxylase; Cell Blotting Technology, USA), anti-DNA-PKcs, anti-phosphate-DNA-PKcs (Abcam, USA) and anti-α-tubulin antibodies were used for immunoblotting.

As a result, as shown in Figure 2a was confirmed that glucose consumption does not affect the expression level and DNA-PKcs expression and phosphorylation of AMPKα. However, AMPK phosphorylation induced by glucose depletion was inhibited in human glioma cells lacking DNA-PKcs. In addition, phosphorylation of ACC at Ser79 was higher in M059K cells than in M059J cells.

<3-2> AMPK Phosphorylation According to Glucose Concentration

M059K and M059J human glioma cells incubated according to the method of Example 2 for 12 hours in a medium containing 17.5, 10, 1 and 0 mM glucose were washed with phosphate buffered saline, and then the whole method was carried out by the method of Example 3-1. After protein extraction, immunoblotting was performed using anti-phosphate-AMPKα, anti-AMPKα, anti-phosphate-ACC and anti-α-tubulin antibodies.

As a result, as shown in Figure 2b, the expression level of AMPKα was maintained constant, but in the absence of glucose was larger in M059K cells than phosphorylation of AMPK and MO59J cells. In addition, phosphorylation of ACC at Ser79 was higher in M059K cells than in M059J cells.

Example 4 Confirmation of AMPKγ1 Activity in Cells Lacking DNA-PKcs

The M059K cell line rich in DNA-PKcs and the M059J cell line deficient in M059K DNA-PKcs were measured for AMPK activity induced by glucose depletion.

1) M059K and M059J human glioma cells in which M059J and M059K cells were cultured in medium with or without glucose for 12 hours were washed with phosphate buffered saline, and then the protein was washed with the method of Example 3-1. After extraction it was quantified. Whole cell ground liquid containing 200 μg of protein was incubated with protein G-Sepharose beads (Amersham Biosciences, USA) and anti-AMPKα antibody at 4 ° C. for 2 hours. The beads were then pelleted by centrifugation at 12,000 rpm for 20 seconds. Carefully remove the supernatant and wash the beads once with a buffer containing 1% Triton X-100, then twice with a buffer without 1% Triton X-100. The beads were suspended in 50 μl of the buffer and AMPK activity was measured.

2) AMPK activity was determined using SAMS (SEQ ID NO 3: HMRSAMSGLHLVKRR) using 5 μl of 5 μCi [γ- ³² P] ATP (IZOTOP, Hungary), 5 μl of AMP (1 mM) and 5 μl of cell lysate assay buffer. 5 μg of synthetic peptide substrate (1 μg / μl; Upstate, USA) was determined by measuring the binding of ³² P (Davies SP et al ., Eur J Biochem 186: 123-128, 1989). After the test mixture was incubated at 30 ° C. for 10 minutes, the reaction was terminated by dropping the mixture on P-81 phosphocellulose paper (Upstate Cell signaling, USA) and washing three times with 1% phosphoric acid three times. After drying the sample, the amount of labeled phosphoric acid binding was quantified using a liquid scintillation counter (Packard Instruments, USA). The test was performed three times. Each figure is expressed as mean ± sd (**, P <0.01 and *, p <0.05).

As a result, as shown in FIG. 3, AMPK activity induced by glucose consumption was suppressed in cells lacking DNA-PK. The baseline AMPK activity of M059J and M059K cells was 995 ± 22 and 1020 ± 25 cpm, respectively, and AMPK activity was increased to 1160 ± 26 and 1480 ± 36 cpm, respectively, in M059J and M059K cells exposed to glucose depletion.

< Example  5> DNA - PK siRNA On by AMPK Confirmation of γ1 Activity

The activity of AMPKγ1 by DNA-PK siRNA was measured in M059K cell line rich in DNA-PKcs.

<5-1> Inhibition of AMPK Phosphorylation by DNA-PK siRNA

1) Using Lipofectamine RNAiMAX (Invitrogen, USA) on M059K cells, Scrambled siRNA (negative control: Bioneer, Korea; Sense siRNA SEQ ID NO: 3: 5'-CCU ACG CCA CCA AUU UCG U-3) according to the manufacturer's protocol ', Antisense siRNA SEQ ID NO: 4: 5'-ACG AAA UUG GUG GCG UAG G-3') or DNA-PKcs siRNA (Santa Cruz Biotechnology, USA: Catalog # SC-35200, nucleotide sequence not disclosed by the company) Transduced.

2) After culturing the transduced cells in medium with or without glucose for 12 hours, the cells were washed with phosphate buffered saline, and the whole protein was extracted by the method of Example 3-1, followed by anti-phosphate-AMPKα. Immunoblotting was performed using anti-AMPKα, anti-DNA-PKcs and anti-α-tubulin antibodies.

As a result, as shown in Figure 4a, in M059K cells transformed with DNA-PKcs siRNA, it was confirmed that the amount of protein expression of DNA-PKcs decreased by more than 80%. In addition, it was confirmed by RT-PCR that the mRNA amount of DNA-PKcs was significantly reduced (data not shown). In addition, it was confirmed that phosphorylation of AMPK was also significantly reduced.

<5-2> Inhibition of AMPK Activity by DNA-PK siRNA

The transduced cells of Example 5-1 were incubated for 12 hours in medium with or without glucose, and then protein isolation, AMPK immunoprecipitation and SAMS peptide analysis were performed by the method of Example 4-1. Each experiment was performed three times separately and each value was presented in the form of mean ± s.d. (**, P <0.01).

As a result, as shown in FIG. 4B, AMPK activity in cells transfected with the control siRNA was increased by approximately 46% (1010 ± 22 vs. 1470 ± 31) by glucose consumption, while the expression of DNA-PKcs under the same conditions. AMPK activity increased only 24% (820 ± 19 vs. 1020 ± 27) in these inhibited cells (see FIG. 4B). The results indicate that DNA-PKcs increases AMPK activity induced by glucose consumption.

<5-3> Inhibition of AMPK Activity by DNA-PK Inhibitor

When culturing M059K cells in glucose-free medium, 20 μM of Waltmannin (Wellmannin; Cell Signaling Technology, USA) was added to inhibit the activity of DNA-PKcs for 1 hour. Cells incubated in glucose-free medium for additional 0, 6, 12 and 24 hours were washed with phosphate buffered saline, and then the whole protein was extracted by the method of Example 3-1, and anti-phosphate-AMPKα, anti- Immunoblotting was performed using AMPKα, anti-phosphate-ACC and anti-α-tubulin antibodies.

As a result, as shown in Figure 4c it was confirmed that in the cells treated with Waltmanin than in the cells not treated with Waltmanin, AMPK phosphorylation stimulated by glucose consumption was increased less. The results indicate that DNA-PKcs contributes to the phosphorylation of AMPK in glucose depleted state.

Example 6 Identification of Inhibition of AMPK Activity in Primary Human Diploid Fibroblasts

Primary human diploid fibroblasts IMR90 and WI38 (ATCC, USA) were cultured in Earle's MEM medium (Invitrogen, USA) containing 10% FBS, penicillin (100 U / ml) and streptomycin (100 μg / ml) In accordance with the method of Example 5-1 was transduced with a control siRNA or DNA-PKcs siRNA. After 48 hours, the transfected cells were further cultured for 0, 6, 12 and 24 hours in glucose free medium and then washed with phosphate buffered saline. Subsequently, the whole protein was extracted by the method of Example 3-1, and immunized using anti-DNA-PKcs, anti-phosphate-AMPKα, anti-AMPKα, anti-phosphate-ACC and anti-α-tubulin antibody. Lotting was performed.

As a result, as shown in FIG. 5, the expression level of DNA-PKcs protein was decreased by inhibiting DNA-PKcs expression in human fibroblast WI38 and IMR90 cell lines, and AMPKα phosphorylation induced by glucose consumption was also reduced. In addition, the amount of ACC phosphorylation in response to glucose consumption was reduced in cells transfected with DNA-PKcs siRNA compared to the control.

Example 7 Confirmation of Inhibition of AMPK Activity in HeLa Cell Line

<7-1> Inhibition of AMPK Activity by DNA-PKcs siRNA

Human cervical cancer cell line HeLa (ATCC, USA) incubated in DMEM (Invitrogen, USA) containing 10% FBS, penicillin (100 U / ml) and streptomycin (100 μg / ml) was subjected to the method of Example 5-1. According to control siRNA or DNA-PKcs siRNA. After 48 hours, the transfected cells were further cultured for 0, 6, 12 and 24 hours in glucose free medium and then washed with phosphate buffered saline. Thereafter, the whole protein was extracted by the method of Example 3-1, and immunoblotting was performed using anti-DNA-PKcs, anti-phosphate-AMPKα, anti-AMPKα, and anti-α-tubulin antibody.

As a result, as shown in Figure 6a, under glucose consumption conditions, phosphorylation of AMPK was significantly reduced in HeLa cells inhibited in the expression of DNA-PKcs compared to control cells.

<7-2> Inhibition of AMPK Activity by DNA-PK Inhibitor

When HeLa cells were cultured in a glucose-free medium, 20 μM of Waltmannin was added to inhibit the activity of DNA-PKcs for 1 hour. Cells incubated in glucose-free medium for additional 0, 6, 12 and 24 hours were washed with phosphate buffered saline, and then the whole protein was extracted by the method of Example 3-1, and anti-phosphate-AMPKα, anti- Immunoblotting was performed using AMPKα and anti-α-tubulin antibodies.

As a result, as shown in FIG. 6B, phosphorylation of AMPK was significantly lower in Waltmannin-treated cells than in untreated control cells.

<7-2> Inhibition of AMPK Activity by CAMKK Inhibitor

When HeLa cells were cultured in glucose-free medium, CAMKK inhibitor STO-609 (1 μg / ml: Sigma, USA) or 20 μM of Waltmannin was added and treated for 6 hours or 1 hour, respectively. Cells incubated in glucose-free medium for an additional 12 hours were washed with phosphate buffered saline, and then the whole protein was extracted by the method of Example 3-1, and anti-phosphate-AMPKα, anti-AMPKα and anti-α- Immunoblotting was performed using tubulin antibodies.

As a result, as shown in Figure 6c it was confirmed that the inhibitory effect of AMPK phosphorylation by glucose consumption is almost the same in Waltmanin and STO-609.

1 is a diagram showing the binding of DNA-PKcs and AMPKγ1 in glioma cells:

a: M059K cell line rich in DNA-PKcs and M059J cell line deficient in M059K DNA-PKcs; And,

b: M059J cell line.

Figure 2 shows that DNA-PKcs promotes AMPKγ1 phosphorylation by glucose consumption:

a: glucose consumption hourly; And,

b: by glucose concentration;

3 is a diagram confirming that DNA-PKcs affects AMPKγ1 activity by glucose consumption.

4 is a diagram confirming that AMPKγ1 phosphorylation and activity and phosphorylation of ACC, a substrate of AMPK, were inhibited in cells in which DNA-PKcs expression and activity were inhibited:

a: AMPKγ1 phosphorylation;

b: AMPKγ1 activity; And,

c: ACC phosphorylation.

5 shows that AMPKγ1 phosphorylation and ACC phosphorylation are inhibited by DNA-PKcs expression inhibition in human fibroblasts.

Figure 6 confirms that AMPKγ1 phosphorylation was inhibited by inhibition of DNA-PKcs expression and activity in HeLa cell lines that lack LKB1:

a: cell in which AMPKγ1 activity was inhibited by siRNA;

b: cells whose AMPKγ1 activity was inhibited by Waltmannin; And,

c: Cells whose AMPKγ1 activity was inhibited by STO-609 and Waltmannin.

<110> Industry Academy Cooperation Foundation Chosun University <120> Method for screening inhibitor of AMPK activity <130> 8P-12-51 <160> 9 <170> KopatentIn 1.71 <210> 1 <211> 331 <212> PRT <213> human AMPK gamma1 <400> 1 Met Glu Thr Val Ile Ser Ser Asp Ser Ser Pro Ala Val Glu Asn Glu   1 5 10 15 His Pro Gln Glu Thr Pro Glu Ser Asn Asn Ser Val Tyr Thr Ser Phe              20 25 30 Met Lys Ser His Arg Cys Tyr Asp Leu Ile Pro Thr Ser Ser Lys Leu          35 40 45 Val Val Phe Asp Thr Ser Leu Gln Val Lys Lys Ala Phe Phe Ala Leu      50 55 60 Val Thr Asn Gly Val Arg Ala Ala Pro Leu Trp Asp Ser Lys Lys Gln  65 70 75 80 Ser Phe Val Gly Met Leu Thr Ile Thr Asp Phe Ile Asn Ile Leu His                  85 90 95 Arg Tyr Tyr Lys Ser Ala Leu Val Gln Ile Tyr Glu Leu Glu Glu His             100 105 110 Lys Ile Glu Thr Trp Arg Glu Val Tyr Leu Gln Asp Ser Phe Lys Pro         115 120 125 Leu Val Cys Ile Ser Pro Asn Ala Ser Leu Phe Asp Ala Val Ser Ser     130 135 140 Leu Ile Arg Asn Lys Ile His Arg Leu Pro Val Ile Asp Pro Glu Ser 145 150 155 160 Gly Asn Thr Leu Tyr Ile Leu Thr His Lys Arg Ile Leu Lys Phe Leu                 165 170 175 Lys Leu Phe Ile Thr Glu Phe Pro Lys Pro Glu Phe Met Ser Lys Ser             180 185 190 Leu Glu Glu Leu Gln Ile Gly Thr Tyr Ala Asn Ile Ala Met Val Arg         195 200 205 Thr Thr Thr Pro Val Tyr Val Ala Leu Gly Ile Phe Val Gln His Arg     210 215 220 Val Ser Ala Leu Pro Val Val Asp Glu Lys Gly Arg Val Val Asp Ile 225 230 235 240 Tyr Ser Lys Phe Asp Val Ile Asn Leu Ala Ala Glu Lys Thr Tyr Asn                 245 250 255 Asn Leu Asp Val Ser Val Thr Lys Ala Leu Gln His Arg Ser His Tyr             260 265 270 Phe Glu Gly Val Leu Lys Cys Tyr Leu His Glu Thr Leu Glu Thr Ile         275 280 285 Ile Asn Arg Leu Val Glu Ala Glu Val His Arg Leu Val Val Val Asp     290 295 300 Glu Asn Asp Val Val Lys Gly Ile Val Ser Leu Ser Asp Ile Leu Gln 305 310 315 320 Ala Leu Val Leu Thr Gly Gly Glu Lys Lys Pro                 325 330 <210> 2 <211> 4128 <212> PRT <213> human DNA-PKcs <400> 2 Met Ala Gly Ser Gly Ala Gly Val Arg Cys Ser Leu Leu Arg Leu Gln   1 5 10 15 Glu Thr Leu Ser Ala Ala Asp Arg Cys Gly Ala Ala Leu Ala Gly His              20 25 30 Gln Leu Ile Arg Gly Leu Gly Gln Glu Cys Val Leu Ser Ser Ser Pro          35 40 45 Ala Val Leu Ala Leu Gln Thr Ser Leu Val Phe Ser Arg Asp Phe Gly      50 55 60 Leu Leu Val Phe Val Arg Lys Ser Leu Asn Ser Ile Glu Phe Arg Glu  65 70 75 80 Cys Arg Glu Glu Ile Leu Lys Phe Leu Cys Ile Phe Leu Glu Lys Met                  85 90 95 Gly Gln Lys Ile Ala Pro Tyr Ser Val Glu Ile Lys Asn Thr Cys Thr             100 105 110 Ser Val Tyr Thr Lys Asp Arg Ala Ala Lys Cys Lys Ile Pro Ala Leu         115 120 125 Asp Leu Leu Ile Lys Leu Leu Gln Thr Phe Arg Ser Ser Arg Leu Met     130 135 140 Asp Glu Phe Lys Ile Gly Glu Leu Phe Ser Lys Phe Tyr Gly Glu Leu 145 150 155 160 Ala Leu Lys Lys Lys Ile Pro Asp Thr Val Leu Glu Lys Val Tyr Glu                 165 170 175 Leu Leu Gly Leu Leu Gly Glu Val His Pro Ser Glu Met Ile Asn Asn             180 185 190 Ala Glu Asn Leu Phe Arg Ala Phe Leu Gly Glu Leu Lys Thr Gln Met         195 200 205 Thr Ser Ala Val Arg Glu Pro Lys Leu Pro Val Leu Ala Gly Cys Leu     210 215 220 Lys Gly Leu Ser Ser Leu Leu Cys Asn Phe Thr Lys Ser Met Glu Glu 225 230 235 240 Asp Pro Gln Thr Ser Arg Glu Ile Phe Asn Phe Val Leu Lys Ala Ile                 245 250 255 Arg Pro Gln Ile Asp Leu Lys Arg Tyr Ala Val Pro Ser Ala Gly Leu             260 265 270 Arg Leu Phe Ala Leu His Ala Ser Gln Phe Ser Thr Cys Leu Leu Asp         275 280 285 Asn Tyr Val Ser Leu Phe Glu Val Leu Leu Lys Trp Cys Ala His Thr     290 295 300 Asn Val Glu Leu Lys Lys Ala Ala Leu Ser Ala Leu Glu Ser Phe Leu 305 310 315 320 Lys Gln Val Ser Asn Met Val Ala Lys Asn Ala Glu Met His Lys Asn                 325 330 335 Lys Leu Gln Tyr Phe Met Glu Gln Phe Tyr Gly Ile Ile Arg Asn Val             340 345 350 Asp Ser Asn Asn Lys Glu Leu Ser Ile Ala Ile Arg Gly Tyr Gly Leu         355 360 365 Phe Ala Gly Pro Cys Lys Val Ile Asn Ala Lys Asp Val Asp Phe Met     370 375 380 Tyr Val Glu Leu Ile Gln Arg Cys Lys Gln Met Phe Leu Thr Gln Thr 385 390 395 400 Asp Thr Gly Asp Asp Arg Val Tyr Gln Met Pro Ser Phe Leu Gln Ser                 405 410 415 Val Ala Ser Val Leu Leu Tyr Leu Asp Thr Val Pro Glu Val Tyr Thr             420 425 430 Pro Val Leu Glu His Leu Val Val Met Gln Ile Asp Ser Phe Pro Gln         435 440 445 Tyr Ser Pro Lys Met Gln Leu Val Cys Cys Arg Ala Ile Val Lys Val     450 455 460 Phe Leu Ala Leu Ala Ala Lys Gly Pro Val Leu Arg Asn Cys Ile Ser 465 470 475 480 Thr Val Val His Gln Gly Leu Ile Arg Ile Cys Ser Lys Pro Val Val                 485 490 495 Leu Pro Lys Gly Pro Glu Ser Glu Ser Glu Asp His Arg Ala Ser Gly             500 505 510 Glu Val Arg Thr Gly Lys Trp Lys Val Pro Thr Tyr Lys Asp Tyr Val         515 520 525 Asp Leu Phe Arg His Leu Leu Ser Ser Asp Gln Met Met Asp Ser Ile     530 535 540 Leu Ala Asp Glu Ala Phe Phe Ser Val Asn Ser Ser Ser Glu Ser Leu 545 550 555 560 Asn His Leu Leu Tyr Asp Glu Phe Val Lys Ser Val Leu Lys Ile Val                 565 570 575 Glu Lys Leu Asp Leu Thr Leu Glu Ile Gln Thr Val Gly Glu Gln Glu             580 585 590 Asn Gly Asp Glu Ala Pro Gly Val Trp Met Ile Pro Thr Ser Asp Pro         595 600 605 Ala Ala Asn Leu His Pro Ala Lys Pro Lys Asp Phe Ser Ala Phe Ile     610 615 620 Asn Leu Val Glu Phe Cys Arg Glu Ile Leu Pro Glu Lys Gln Ala Glu 625 630 635 640 Phe Phe Glu Pro Trp Val Tyr Ser Phe Ser Tyr Glu Leu Ile Leu Gln                 645 650 655 Ser Thr Arg Leu Pro Leu Ile Ser Gly Phe Tyr Lys Leu Leu Ser Ile             660 665 670 Thr Val Arg Asn Ala Lys Lys Ile Lys Tyr Phe Glu Gly Val Ser Pro         675 680 685 Lys Ser Leu Lys His Ser Pro Glu Asp Pro Glu Lys Tyr Ser Cys Phe     690 695 700 Ala Leu Phe Val Lys Phe Gly Lys Glu Val Ala Val Lys Met Lys Gln 705 710 715 720 Tyr Lys Asp Glu Leu Leu Ala Ser Cys Leu Thr Phe Leu Leu Ser Leu                 725 730 735 Pro His Asn Ile Ile Glu Leu Asp Val Arg Ala Tyr Val Pro Ala Leu             740 745 750 Gln Met Ala Phe Lys Leu Gly Leu Ser Tyr Thr Pro Leu Ala Glu Val         755 760 765 Gly Leu Asn Ala Leu Glu Glu Trp Ser Ile Tyr Ile Asp Arg His Val     770 775 780 Met Gln Pro Tyr Tyr Lys Asp Ile Leu Pro Cys Leu Asp Gly Tyr Leu 785 790 795 800 Lys Thr Ser Ala Leu Ser Asp Glu Thr Lys Asn Asn Trp Glu Val Ser                 805 810 815 Ala Leu Ser Arg Ala Ala Gln Lys Gly Phe Asn Lys Val Val Leu Lys             820 825 830 His Leu Lys Lys Thr Lys Asn Leu Ser Ser Asn Glu Ala Ile Ser Leu         835 840 845 Glu Glu Ile Arg Ile Arg Val Val Gln Met Leu Gly Ser Leu Gly Gly     850 855 860 Gln Ile Asn Lys Asn Leu Leu Thr Val Thr Ser Ser Asp Glu Met Met 865 870 875 880 Lys Ser Tyr Val Ala Trp Asp Arg Glu Lys Arg Leu Ser Phe Ala Val                 885 890 895 Pro Phe Arg Glu Met Lys Pro Val Ile Phe Leu Asp Val Phe Leu Pro             900 905 910 Arg Val Thr Glu Leu Ala Leu Thr Ala Ser Asp Arg Gln Thr Lys Val         915 920 925 Ala Ala Cys Glu Leu Leu His Ser Met Val Met Phe Met Leu Gly Lys     930 935 940 Ala Thr Gln Met Pro Glu Gly Gly Gln Gly Ala Pro Pro Met Tyr Gln 945 950 955 960 Leu Tyr Lys Arg Thr Phe Pro Val Leu Leu Arg Leu Ala Cys Asp Val                 965 970 975 Asp Gln Val Thr Arg Gln Leu Tyr Glu Pro Leu Val Met Gln Leu Ile             980 985 990 His Trp Phe Thr Asn Asn Lys Lys Phe Glu Ser Gln Asp Thr Val Ala         995 1000 1005 Leu Leu Glu Ala Ile Leu Asp Gly Ile Val Asp Pro Val Asp Ser Thr    1010 1015 1020 Leu Arg Asp Phe Cys Gly Arg Cys Ile Arg Glu Phe Leu Lys Trp Ser 1025 1030 1035 1040 Ile Lys Gln Ile Thr Pro Gln Gln Gln Glu Lys Ser Pro Val Asn Thr                1045 1050 1055 Lys Ser Leu Phe Lys Arg Leu Tyr Ser Leu Ala Leu His Pro Asn Ala            1060 1065 1070 Phe Lys Arg Leu Gly Ala Ser Leu Ala Phe Asn Asn Ile Tyr Arg Glu        1075 1080 1085 Phe Arg Glu Glu Glu Ser Leu Val Glu Gln Phe Val Phe Glu Ala Leu    1090 1095 1100 Val Ile Tyr Met Glu Ser Leu Ala Leu Ala His Ala Asp Glu Lys Ser 1105 1110 1115 1120 Leu Gly Thr Ile Gln Gln Cys Cys Asp Ala Ile Asp His Leu Cys Arg                1125 1130 1135 Ile Ile Glu Lys Lys His Val Ser Leu Asn Lys Ala Lys Lys Arg Arg            1140 1145 1150 Leu Pro Arg Gly Phe Pro Pro Ser Ala Ser Leu Cys Leu Leu Asp Leu        1155 1160 1165 Val Lys Trp Leu Leu Ala His Cys Gly Arg Pro Gln Thr Glu Cys Arg    1170 1175 1180 His Lys Ser Ile Glu Leu Phe Tyr Lys Phe Val Pro Leu Leu Pro Gly 1185 1190 1195 1200 Asn Arg Ser Pro Asn Leu Trp Leu Lys Asp Val Leu Lys Glu Glu Gly                1205 1210 1215 Val Ser Phe Leu Ile Asn Thr Phe Glu Gly Gly Gly Cys Gly Gln Pro            1220 1225 1230 Ser Gly Ile Leu Ala Gln Pro Thr Leu Leu Tyr Leu Arg Gly Pro Phe        1235 1240 1245 Ser Leu Gln Ala Thr Leu Cys Trp Leu Asp Leu Leu Leu Ala Ala Leu    1250 1255 1260 Glu Cys Tyr Asn Thr Phe Ile Gly Glu Arg Thr Val Gly Ala Leu Gln 1265 1270 1275 1280 Val Leu Gly Thr Glu Ala Gln Ser Ser Leu Leu Lys Ala Val Ala Phe                1285 1290 1295 Phe Leu Glu Ser Ile Ala Met His Asp Ile Ile Ala Ala Glu Lys Cys            1300 1305 1310 Phe Gly Thr Gly Ala Ala Gly Asn Arg Thr Ser Pro Gln Glu Gly Glu        1315 1320 1325 Arg Tyr Asn Tyr Ser Lys Cys Thr Val Val Val Arg Ile Met Glu Phe    1330 1335 1340 Thr Thr Thr Le Leu Leu Asn Thr Ser Pro Glu Gly Trp Lys Leu Leu Lys 1345 1350 1355 1360 Lys Asp Leu Cys Asn Thr His Leu Met Arg Val Leu Val Gln Thr Leu                1365 1370 1375 Cys Glu Pro Ala Ser Ile Gly Phe Asn Ile Gly Asp Val Gln Val Met            1380 1385 1390 Ala His Leu Pro Asp Val Cys Val Asn Leu Met Lys Ala Leu Lys Met        1395 1400 1405 Ser Pro Tyr Lys Asp Ile Leu Glu Thr His Leu Arg Glu Lys Ile Thr    1410 1415 1420 Ala Gln Ser Ile Glu Glu Leu Cys Ala Val Asn Leu Tyr Gly Pro Asp 1425 1430 1435 1440 Ala Gln Val Asp Arg Ser Arg Leu Ala Ala Val Val Ser Ala Cys Lys                1445 1450 1455 Gln Leu His Arg Ala Gly Leu Leu His Asn Ile Leu Pro Ser Gln Ser            1460 1465 1470 Thr Asp Leu His His Ser Val Gly Thr Glu Leu Leu Ser Leu Val Tyr        1475 1480 1485 Lys Gly Ile Ala Pro Gly Asp Glu Arg Gln Cys Leu Pro Ser Leu Asp    1490 1495 1500 Leu Ser Cys Lys Gln Leu Ala Ser Gly Leu Leu Glu Leu Ala Phe Ala 1505 1510 1515 1520 Phe Gly Gly Leu Cys Glu Arg Leu Val Ser Leu Leu Leu Asn Pro Ala                1525 1530 1535 Val Leu Ser Thr Ala Ser Leu Gly Ser Ser Gln Gly Ser Val Ile His            1540 1545 1550 Phe Ser His Gly Glu Tyr Phe Tyr Ser Leu Phe Ser Glu Thr Ile Asn        1555 1560 1565 Thr Glu Leu Leu Lys Asn Leu Asp Leu Ala Val Leu Glu Leu Met Gln    1570 1575 1580 Ser Ser Val Asp Asn Thr Lys Met Val Ser Ala Val Leu Asn Gly Met 1585 1590 1595 1600 Leu Asp Gln Ser Phe Arg Glu Arg Ala Asn Gln Lys His Gln Gly Leu                1605 1610 1615 Lys Leu Ala Thr Thr Ile Leu Gln His Trp Lys Lys Cys Asp Ser Trp            1620 1625 1630 Trp Ala Lys Asp Ser Pro Leu Glu Thr Lys Met Ala Val Leu Ala Leu        1635 1640 1645 Leu Ala Lys Ile Leu Gln Ile Asp Ser Ser Val Ser Phe Asn Thr Ser    1650 1655 1660 His Gly Ser Phe Pro Glu Val Phe Thr Thr Tyr Ile Ser Leu Leu Ala 1665 1670 1675 1680 Asp Thr Lys Leu Asp Leu His Leu Lys Gly Gln Ala Val Thr Leu Leu                1685 1690 1695 Pro Phe Phe Thr Ser Leu Thr Gly Gly Ser Leu Glu Glu Leu Arg Arg            1700 1705 1710 Val Leu Glu Gln Leu Ile Val Ala His Phe Pro Met Gln Ser Arg Glu        1715 1720 1725 Phe Pro Pro Gly Thr Pro Arg Phe Asn Asn Tyr Val Asp Cys Met Lys    1730 1735 1740 Lys Phe Leu Asp Ala Leu Glu Leu Ser Gln Ser Pro Met Leu Leu Glu 1745 1750 1755 1760 Leu Met Thr Glu Val Leu Cys Arg Glu Gln Gln His Val Met Glu Glu                1765 1770 1775 Leu Phe Gln Ser Ser Phe Arg Arg Ile Ala Arg Arg Gly Ser Cys Val            1780 1785 1790 Thr Gln Val Gly Leu Leu Glu Ser Val Tyr Glu Met Phe Arg Lys Asp        1795 1800 1805 Asp Pro Arg Leu Ser Phe Thr Arg Gln Ser Phe Val Asp Arg Ser Leu    1810 1815 1820 Leu Thr Leu Leu Trp His Cys Ser Leu Asp Ala Leu Arg Glu Phe Phe 1825 1830 1835 1840 Ser Thr Ile Val Val Asp Ala Ile Asp Val Leu Lys Ser Arg Phe Thr                1845 1850 1855 Lys Leu Asn Glu Ser Thr Phe Asp Thr Gln Ile Thr Lys Lys Met Gly            1860 1865 1870 Tyr Tyr Lys Ile Leu Asp Val Met Tyr Ser Arg Leu Pro Lys Asp Asp        1875 1880 1885 Val His Ala Lys Glu Ser Lys Ile Asn Gln Val Phe His Gly Ser Cys    1890 1895 1900 Ile Thr Glu Gly Asn Glu Leu Thr Lys Thr Leu Ile Lys Leu Cys Tyr 1905 1910 1915 1920 Asp Ala Phe Thr Glu Asn Met Ala Gly Glu Asn Gln Leu Leu Glu Arg                1925 1930 1935 Arg Arg Leu Tyr His Cys Ala Ala Tyr Asn Cys Ala Ile Ser Val Ile            1940 1945 1950 Cys Cys Val Phe Asn Glu Leu Lys Phe Tyr Gln Gly Phe Leu Phe Ser        1955 1960 1965 Glu Lys Pro Glu Lys Asn Leu Leu Ile Phe Glu Asn Leu Ile Asp Leu    1970 1975 1980 Lys Arg Arg Tyr Asn Phe Pro Val Glu Val Glu Val Pro Met Glu Arg 1985 1990 1995 2000 Lys Lys Lys Tyr Ile Glu Ile Arg Lys Glu Ala Arg Glu Ala Ala Asn                2005 2010 2015 Gly Asp Ser Asp Gly Pro Ser Tyr Met Ser Ser Leu Ser Tyr Leu Ala            2020 2025 2030 Asp Ser Thr Leu Ser Glu Glu Met Ser Gln Phe Asp Phe Ser Thr Gly        2035 2040 2045 Val Gln Ser Tyr Ser Tyr Ser Ser Gln Asp Pro Arg Pro Ala Thr Gly    2050 2055 2060 Arg Phe Arg Arg Arg Glu Gln Arg Asp Pro Thr Val His Asp Asp Val 2065 2070 2075 2080 Leu Glu Leu Glu Met Asp Glu Leu Asn Arg His Glu Cys Met Ala Pro                2085 2090 2095 Leu Thr Ala Leu Val Lys His Met His Arg Ser Leu Gly Pro Pro Gln            2100 2105 2110 Gly Glu Glu Asp Ser Val Pro Arg Asp Leu Pro Ser Trp Met Lys Phe        2115 2120 2125 Leu His Gly Lys Leu Gly Asn Pro Ile Val Pro Leu Asn Ile Arg Leu    2130 2135 2140 Phe Leu Ala Lys Leu Val Ile Asn Thr Glu Glu Val Phe Arg Pro Tyr 2145 2150 2155 2160 Ala Lys His Trp Leu Ser Pro Leu Leu Gln Leu Ala Ala Ser Glu Asn                2165 2170 2175 Asn Gly Gly Glu Gly Ile His Tyr Met Val Val Glu Ile Val Ala Thr            2180 2185 2190 Ile Leu Ser Trp Thr Gly Leu Ala Thr Pro Thr Gly Val Pro Lys Asp        2195 2200 2205 Glu Val Leu Ala Asn Arg Leu Leu Asn Phe Leu Met Lys His Val Phe    2210 2215 2220 His Pro Lys Arg Ala Val Phe Arg His Asn Leu Glu Ile Ile Lys Thr 2225 2230 2235 2240 Leu Val Glu Cys Trp Lys Asp Cys Leu Ser Ile Pro Tyr Arg Leu Ile                2245 2250 2255 Phe Glu Lys Phe Ser Gly Lys Asp Pro Asn Ser Lys Asp Asn Ser Val            2260 2265 2270 Gly Ile Gln Leu Leu Gly Ile Val Met Ala Asn Asp Leu Pro Pro Tyr        2275 2280 2285 Asp Pro Gln Cys Gly Ile Gln Ser Ser Glu Tyr Phe Gln Ala Leu Val    2290 2295 2300 Asn Asn Met Ser Phe Val Arg Tyr Lys Glu Val Tyr Ala Ala Ala Ala 2305 2310 2315 2320 Glu Val Leu Gly Leu Ile Leu Arg Tyr Val Met Glu Arg Lys Asn Ile                2325 2330 2335 Leu Glu Glu Ser Leu Cys Glu Leu Val Ala Lys Gln Leu Lys Gln His            2340 2345 2350 Gln Asn Thr Met Glu Asp Lys Phe Ile Val Cys Leu Asn Lys Val Thr        2355 2360 2365 Lys Ser Phe Pro Pro Leu Ala Asp Arg Phe Met Asn Ala Val Phe Phe    2370 2375 2380 Leu Leu Pro Lys Phe His Gly Val Leu Lys Thr Leu Cys Leu Glu Val 2385 2390 2395 2400 Val Leu Cys Arg Val Glu Gly Met Thr Glu Leu Tyr Phe Gln Leu Lys                2405 2410 2415 Ser Lys Asp Phe Val Gln Val Met Arg His Arg Asp Asp Glu Arg Gln            2420 2425 2430 Lys Val Cys Leu Asp Ile Ile Tyr Lys Met Met Pro Lys Leu Lys Pro        2435 2440 2445 Val Glu Leu Arg Glu Leu Leu Asn Pro Val Val Glu Phe Val Ser His    2450 2455 2460 Pro Ser Thr Thr Cys Arg Glu Gln Met Tyr Asn Ile Leu Met Trp Ile 2465 2470 2475 2480 His Asp Asn Tyr Arg Asp Pro Glu Ser Glu Thr Asp Asn Asp Ser Gln                2485 2490 2495 Glu Ile Phe Lys Leu Ala Lys Asp Val Leu Ile Gln Gly Leu Ile Asp            2500 2505 2510 Glu Asn Pro Gly Leu Gln Leu Ile Ile Arg Asn Phe Trp Ser His Glu        2515 2520 2525 Thr Arg Leu Pro Ser Asn Thr Leu Asp Arg Leu Leu Ala Leu Asn Ser    2530 2535 2540 Leu Tyr Ser Pro Lys Ile Glu Val His Phe Leu Ser Leu Ala Thr Asn 2545 2550 2555 2560 Phe Leu Leu Glu Met Thr Ser Met Ser Pro Asp Tyr Pro Asn Pro Met                2565 2570 2575 Phe Glu His Pro Leu Ser Glu Cys Glu Phe Gln Glu Tyr Thr Ile Asp            2580 2585 2590 Ser Asp Trp Arg Phe Arg Ser Thr Val Leu Thr Pro Met Phe Val Glu        2595 2600 2605 Thr Gln Ala Ser Gln Gly Thr Leu Gln Thr Arg Thr Gln Glu Gly Ser    2610 2615 2620 Leu Ser Ala Arg Trp Pro Val Ala Gly Gln Ile Arg Ala Thr Gln Gln 2625 2630 2635 2640 Gln His Asp Phe Thr Leu Thr Gln Thr Ala Asp Gly Arg Ser Ser Phe                2645 2650 2655 Asp Trp Leu Thr Gly Ser Ser Thr Asp Pro Leu Val Asp His Thr Ser            2660 2665 2670 Pro Ser Ser Asp Ser Leu Leu Phe Ala His Lys Arg Ser Glu Arg Leu        2675 2680 2685 Gln Arg Ala Pro Leu Lys Ser Val Gly Pro Asp Phe Gly Lys Lys Arg    2690 2695 2700 Leu Gly Leu Pro Gly Asp Glu Val Asp Asn Lys Val Lys Gly Ala Ala 2705 2710 2715 2720 Gly Arg Thr Asp Leu Leu Arg Leu Arg Arg Arg Phe Met Arg Asp Gln                2725 2730 2735 Glu Lys Leu Ser Leu Met Tyr Ala Arg Lys Gly Val Ala Glu Gln Lys            2740 2745 2750 Arg Glu Lys Glu Ile Lys Ser Glu Leu Lys Met Lys Gln Asp Ala Gln        2755 2760 2765 Val Val Leu Tyr Arg Ser Tyr Arg His Gly Asp Leu Pro Asp Ile Gln    2770 2775 2780 Ile Lys His Ser Ser Leu Ile Thr Pro Leu Gln Ala Val Ala Gln Arg 2785 2790 2795 2800 Asp Pro Ile Ile Ala Lys Gln Leu Phe Ser Ser Leu Phe Ser Gly Ile                2805 2810 2815 Leu Lys Glu Met Asp Lys Phe Lys Thr Leu Ser Glu Lys Asn Asn Ile            2820 2825 2830 Thr Gln Lys Leu Leu Gln Asp Phe Asn Arg Phe Leu Asn Thr Thr Phe        2835 2840 2845 Ser Phe Phe Pro Pro Phe Val Ser Cys Ile Gln Asp Ile Ser Cys Gln    2850 2855 2860 His Ala Ala Leu Leu Ser Leu Asp Pro Ala Ala Val Ser Ala Gly Cys 2865 2870 2875 2880 Leu Ala Ser Leu Gln Gln Pro Val Gly Ile Arg Leu Leu Glu Glu Ala                2885 2890 2895 Leu Leu Arg Leu Leu Pro Ala Glu Leu Pro Ala Lys Arg Val Arg Gly            2900 2905 2910 Lys Ala Arg Leu Pro Pro Asp Val Leu Arg Trp Val Glu Leu Ala Lys        2915 2920 2925 Leu Tyr Arg Ser Ile Gly Glu Tyr Asp Val Leu Arg Gly Ile Phe Thr    2930 2935 2940 Ser Glu Ile Gly Thr Lys Gln Ile Thr Gln Ser Ala Leu Leu Ala Glu 2945 2950 2955 2960 Ala Arg Ser Asp Tyr Ser Glu Ala Ala Lys Gln Tyr Asp Glu Ala Leu                2965 2970 2975 Asn Lys Gln Asp Trp Val Asp Gly Glu Pro Thr Glu Ala Glu Lys Asp            2980 2985 2990 Phe Trp Glu Leu Ala Ser Leu Asp Cys Tyr Asn His Leu Ala Glu Trp        2995 3000 3005 Lys Ser Leu Glu Tyr Cys Ser Thr Ala Ser Ile Asp Ser Glu Asn Pro    3010 3015 3020 Pro Asp Leu Asn Lys Ile Trp Ser Glu Pro Phe Tyr Gln Glu Thr Tyr 3025 3030 3035 3040 Leu Pro Tyr Met Ile Arg Ser Lys Leu Lys Leu Leu Leu Gln Gly Glu                3045 3050 3055 Ala Asp Gln Ser Leu Leu Thr Phe Ile Asp Lys Ala Met His Gly Glu            3060 3065 3070 Leu Gln Lys Ala Ile Leu Glu Leu His Tyr Ser Gln Glu Leu Ser Leu        3075 3080 3085 Leu Tyr Leu Leu Gln Asp Asp Val Asp Arg Ala Lys Tyr Tyr Ile Gln    3090 3095 3100 Asn Gly Ile Gln Ser Phe Met Gln Asn Tyr Ser Ser Ile Asp Val Leu 3105 3110 3115 3120 Leu His Gln Ser Arg Leu Thr Lys Leu Gln Ser Val Gln Ala Leu Thr                3125 3130 3135 Glu Ile Gln Glu Phe Ile Ser Phe Ile Ser Lys Gln Gly Asn Leu Ser            3140 3145 3150 Ser Gln Val Pro Leu Lys Arg Leu Leu Asn Thr Trp Thr Asn Arg Tyr        3155 3160 3165 Pro Asp Ala Lys Met Asp Pro Met Asn Ile Trp Asp Asp Ile Ile Thr    3170 3175 3180 Asn Arg Cys Phe Phe Leu Ser Lys Ile Glu Glu Lys Leu Thr Pro Leu 3185 3190 3195 3200 Pro Glu Asp Asn Ser Met Asn Val Asp Gln Asp Gly Asp Pro Ser Asp                3205 3210 3215 Arg Met Glu Val Gln Glu Gln Glu Glu Asp Ile Ser Ser Leu Ile Arg            3220 3225 3230 Ser Cys Lys Phe Ser Met Lys Met Lys Met Ile Asp Ser Ala Arg Lys        3235 3240 3245 Gln Asn Asn Phe Ser Leu Ala Met Lys Leu Leu Lys Glu Leu His Lys    3250 3255 3260 Glu Ser Lys Thr Arg Asp Asp Trp Leu Val Ser Trp Val Gln Ser Tyr 3265 3270 3275 3280 Cys Arg Leu Ser His Cys Arg Ser Arg Ser Gln Gly Cys Ser Glu Gln                3285 3290 3295 Val Leu Thr Val Leu Lys Thr Val Ser Leu Leu Asp Glu Asn Asn Val            3300 3305 3310 Ser Ser Tyr Leu Ser Lys Asn Ile Leu Ala Phe Arg Asp Gln Asn Ile        3315 3320 3325 Leu Leu Gly Thr Thr Tyr Arg Ile Ile Ala Asn Ala Leu Ser Ser Glu    3330 3335 3340 Pro Ala Cys Leu Ala Glu Ile Glu Glu Asp Lys Ala Arg Arg Ile Leu 3345 3350 3355 3360 Glu Leu Ser Gly Ser Ser Ser Glu Asp Ser Glu Lys Val Ile Ala Gly                3365 3370 3375 Leu Tyr Gln Arg Ala Phe Gln His Leu Ser Glu Ala Val Gln Ala Ala            3380 3385 3390 Glu Glu Glu Ala Gln Pro Pro Ser Trp Ser Cys Gly Pro Ala Ala Gly        3395 3400 3405 Val Ile Asp Ala Tyr Met Thr Leu Ala Asp Phe Cys Asp Gln Gln Leu    3410 3415 3420 Arg Lys Glu Glu Glu Asn Ala Ser Val Ile Asp Ser Ala Glu Leu Gln 3425 3430 3435 3440 Ala Tyr Pro Ala Leu Val Val Glu Lys Met Leu Lys Ala Leu Lys Leu                3445 3450 3455 Asn Ser Asn Glu Ala Arg Leu Lys Phe Pro Arg Leu Leu Gln Ile Ile            3460 3465 3470 Glu Arg Tyr Pro Glu Glu Thr Leu Ser Leu Met Thr Lys Glu Ile Ser        3475 3480 3485 Ser Val Pro Cys Trp Gln Phe Ile Ser Trp Ile Ser His Met Val Ala    3490 3495 3500 Leu Leu Asp Lys Asp Gln Ala Val Ala Val Gln His Ser Val Glu Glu 3505 3510 3515 3520 Ile Thr Asp Asn Tyr Pro Gln Ala Ile Val Tyr Pro Phe Ile Ile Ser                3525 3530 3535 Ser Glu Ser Tyr Ser Phe Lys Asp Thr Ser Thr Gly His Lys Asn Lys            3540 3545 3550 Glu Phe Val Ala Arg Ile Lys Ser Lys Leu Asp Gln Gly Gly Val Ile        3555 3560 3565 Gln Asp Phe Ile Asn Ala Leu Asp Gln Leu Ser Asn Pro Glu Leu Leu    3570 3575 3580 Phe Lys Asp Trp Ser Asn Asp Val Arg Ala Glu Leu Ala Lys Thr Pro 3585 3590 3595 3600 Val Asn Lys Lys Asn Ile Glu Lys Met Tyr Glu Arg Met Tyr Ala Ala                3605 3610 3615 Leu Gly Asp Pro Lys Ala Pro Gly Leu Gly Ala Phe Arg Arg Lys Phe            3620 3625 3630 Ile Gln Thr Phe Gly Lys Glu Phe Asp Lys His Phe Gly Lys Gly Gly        3635 3640 3645 Ser Lys Leu Leu Arg Met Lys Leu Ser Asp Phe Asn Asp Ile Thr Asn    3650 3655 3660 Met Leu Leu Leu Lys Met Asn Lys Asp Ser Lys Pro Pro Gly Asn Leu 3665 3670 3675 3680 Lys Glu Cys Ser Pro Trp Met Ser Asp Phe Lys Val Glu Phe Leu Arg                3685 3690 3695 Asn Glu Leu Glu Ile Pro Gly Gln Tyr Asp Gly Arg Gly Lys Pro Leu            3700 3705 3710 Pro Glu Tyr His Val Arg Ile Ala Gly Phe Asp Glu Arg Val Thr Val        3715 3720 3725 Met Ala Ser Leu Arg Arg Pro Lys Arg Ile Ile Ile Arg Gly His Asp    3730 3735 3740 Glu Arg Glu His Pro Phe Leu Val Lys Gly Gly Glu Asp Leu Arg Gln 3745 3750 3755 3760 Asp Gln Arg Val Glu Gln Leu Phe Gln Val Met Asn Gly Ile Leu Ala                3765 3770 3775 Gln Asp Ser Ala Cys Ser Gln Arg Ala Leu Gln Leu Arg Thr Tyr Ser            3780 3785 3790 Val Val Pro Met Thr Ser Arg Leu Gly Leu Ile Glu Trp Leu Glu Asn        3795 3800 3805 Thr Val Thr Leu Lys Asp Leu Leu Leu Asn Thr Met Ser Gln Glu Glu    3810 3815 3820 Lys Ala Ala Tyr Leu Ser Asp Pro Arg Ala Pro Pro Cys Glu Tyr Lys 3825 3830 3835 3840 Asp Trp Leu Thr Lys Met Ser Gly Lys His Asp Val Gly Ala Tyr Met                3845 3850 3855 Leu Met Tyr Lys Gly Ala Asn Arg Thr Glu Thr Val Thr Ser Phe Arg            3860 3865 3870 Lys Arg Glu Ser Lys Val Pro Ala Asp Leu Leu Lys Arg Ala Phe Val        3875 3880 3885 Arg Met Ser Thr Ser Pro Glu Ala Phe Leu Ala Leu Arg Ser His Phe    3890 3895 3900 Ala Ser Ser His Ala Leu Ile Cys Ile Ser His Trp Ile Leu Gly Ile 3905 3910 3915 3920 Gly Asp Arg His Leu Asn Asn Phe Met Val Ala Met Glu Thr Gly Gly                3925 3930 3935 Val Ile Gly Ile Asp Phe Gly His Ala Phe Gly Ser Ala Thr Gln Phe            3940 3945 3950 Leu Pro Val Pro Glu Leu Met Pro Phe Arg Leu Thr Arg Gln Phe Ile        3955 3960 3965 Asn Leu Met Leu Pro Met Lys Glu Thr Gly Leu Met Tyr Ser Ile Met    3970 3975 3980 Val His Ala Leu Arg Ala Phe Arg Ser Asp Pro Gly Leu Leu Thr Asn 3985 3990 3995 4000 Thr Met Asp Val Phe Val Lys Glu Pro Ser Phe Asp Trp Lys Asn Phe                4005 4010 4015 Glu Gln Lys Met Leu Lys Lys Gly Gly Ser Trp Ile Gln Glu Ile Asn            4020 4025 4030 Val Ala Glu Lys Asn Trp Tyr Pro Arg Gln Lys Ile Cys Tyr Ala Lys        4035 4040 4045 Arg Lys Leu Ala Gly Ala Asn Pro Ala Val Ile Thr Cys Asp Glu Leu    4050 4055 4060 Leu Leu Gly His Glu Lys Ala Pro Ala Phe Arg Asp Tyr Val Ala Val 4065 4070 4075 4080 Ala Arg Gly Ser Lys Asp His Asn Ile Arg Ala Gln Glu Pro Glu Ser                4085 4090 4095 Gly Leu Ser Glu Glu Thr Gln Val Lys Cys Leu Met Asp Gln Ala Thr            4100 4105 4110 Asp Pro Asn Ile Leu Gly Arg Thr Trp Glu Gly Trp Glu Pro Trp Met        4115 4120 4125 <210> 3 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> SAMS <400> 3 His Met Arg Ser Ala Met Ser Gly Leu His Leu Val Lys Arg Arg   1 5 10 15 <210> 4 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> Scrambled sense siRNA <400> 4 ccuacgccac caauuucgu 19 <210> 5 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> Scrambled antisense siRNA <400> 5 acgaaauugg uggcguagg 19 <210> 6 <211> 13509 <212> DNA <213> human DNA-PKcs <400> 6 ggggcatttc cgggtccggg ccgagcgggc gcacgcgcgg gagcgggact cggcggcatg 60 gcgggctccg gagccggtgt gcgttgctcc ctgctgcggc tgcaggagac cttgtccgct 120 gcggaccgct gcggtgctgc cctggccggt catcaactga tccgcggcct ggggcaggaa 180 tgcgtcctga gcagcagccc cgcggtgctg gcattacaga catctttagt tttttccaga 240 gatttcggtt tgcttgtatt tgtccggaag tcactcaaca gtattgaatt tcgtgaatgt 300 agagaagaaa tcctaaagtt tttatgtatt ttcttagaaa aaatgggcca gaagatcgca 360 ccttactctg ttgaaattaa gaacacttgt accagtgttt atacaaaaga tagagctgct 420 aaatgtaaaa ttccagccct ggaccttctt attaagttac ttcagacttt tagaagttct 480 agactcatgg atgaatttaa aattggagaa ttatttagta aattctatgg agaacttgca 540 ttgaaaaaaa aaataccaga tacagtttta gaaaaagtat atgagctcct aggattattg 600 ggtgaagttc atcctagtga gatgataaat aatgcagaaa acctgttccg cgcttttctg 660 ggtgaactta agacccagat gacatcagca gtaagagagc ccaaactacc tgttctggca 720 ggatgtctga aggggttgtc ctcacttctg tgcaacttca ctaagtccat ggaagaagat 780 ccccagactt caagggagat ttttaatttt gtactaaagg caattcgtcc tcagattgat 840 ctgaagagat atgctgtgcc ctcagctggc ttgcgcctat ttgccctgca tgcatctcag 900 tttagcacct gccttctgga caactacgtg tctctatttg aagtcttgtt aaagtggtgt 960 gcccacacaa atgtagaatt gaaaaaagct gcactttcag ccctggaatc ctttctgaaa 1020 caggtttcta atatggtggc gaaaaatgca gaaatgcata aaaataaact gcagtacttt 1080 atggagcagt tttatggaat catcagaaat gtggattcga acaacaagga gttatctatt 1140 gctatccgtg gatatggact ttttgcagga ccgtgcaagg ttataaacgc aaaagatgtt 1200 gacttcatgt acgttgagct cattcagcgc tgcaagcaga tgttcctcac ccagacagac 1260 actggtgacg accgtgttta tcagatgcca agcttcctcc agtctgttgc aagcgtcttg 1320 ctgtaccttg acacagttcc tgaggtgtat actccagttc tggagcacct cgtggtgatg 1380 cagatagaca gtttcccaca gtacagtcca aaaatgcagc tggtgtgttg cagagccata 1440 gtgaaggtgt tcctagcttt ggcagcaaaa gggccagttc tcaggaattg cattagtact 1500 gtggtgcatc agggtttaat cagaatatgt tctaaaccag tggtccttcc aaagggccct 1560 gagtctgaat ctgaagacca ccgtgcttca ggggaagtca gaactggcaa atggaaggtg 1620 cccacataca aagactacgt ggatctcttc agacatctcc tgagctctga ccagatgatg 1680 gattctattt tagcagatga agcatttttc tctgtgaatt cctccagtga aagtctgaat 1740 catttacttt atgatgaatt tgtaaaatcc gttttgaaga ttgttgagaa attggatctt 1800 acacttgaaa tacagactgt tggggaacaa gagaatggag atgaggcgcc tggtgtttgg 1860 atgatcccaa cttcagatcc agcggctaac ttgcatccag ctaaacctaa agatttttcg 1920 gctttcatta acctggtgga attttgcaga gagattctcc ctgagaaaca agcagaattt 1980 tttgaaccat gggtgtactc attttcatat gaattaattt tgcaatctac aaggttgccc 2040 ctcatcagtg gtttctacaa attgctttct attacagtaa gaaatgccaa gaaaataaaa 2100 tatttcgagg gagttagtcc aaagagtctg aaacactctc ctgaagaccc agaaaagtat 2160 tcttgctttg ctttatttgt gaaatttggc aaagaggtgg cagttaaaat gaagcagtac 2220 aaagatgaac ttttggcctc ttgtttgacc tttcttctgt ccttgccaca caacatcatt 2280 gaactcgatg ttagagccta cgttcctgca ctgcagatgg ctttcaaact gggcctgagc 2340 tataccccct tggcagaagt aggcctgaat gctctagaag aatggtcaat ttatattgac 2400 agacatgtaa tgcagcctta ttacaaagac attctcccct gcctggatgg atacctgaag 2460 acttcagcct tgtcagatga gaccaagaat aactgggaag tgtcagctct ttctcgggct 2520 gcccagaaag gatttaataa agtggtgtta aagcatctga agaagacaaa gaacctttca 2580 tcaaacgaag caatatcctt agaagaaata agaattagag tagtacaaat gcttggatct 2640 ctaggaggac aaataaacaa aaatcttctg acagtcacgt cctcagatga gatgatgaag 2700 agctatgtgg cctgggacag agagaagcgg ctgagctttg cagtgccctt tagagagatg 2760 aaacctgtca ttttcctgga tgtgttcctg cctcgagtca cagaattagc gctcacagcc 2820 agtgacagac aaactaaagt tgcagcctgt gaacttttac atagcatggt tatgtttatg 2880 ttgggcaaag ccacgcagat gccagaaggg ggacagggag ccccacccat gtaccagctc 2940 tataagcgga cgtttcctgt gctgcttcga cttgcgtgtg atgttgatca ggtgacaagg 3000 caactgtatg agccactagt tatgcagctg attcactggt tcactaacaa caagaaattt 3060 gaaagtcagg atactgttgc cttactagaa gctatattgg atggaattgt ggaccctgtt 3120 gacagtactt taagagattt ttgtggtcgg tgtattcgag aattccttaa atggtccatt 3180 aagcaaataa caccacagca gcaggagaag agtccagtaa acaccaaatc gcttttcaag 3240 cgactttata gccttgcgct tcaccccaat gctttcaaga ggctgggagc atcacttgcc 3300 tttaataata tctacaggga attcagggaa gaagagtctc tggtggaaca gtttgtgttt 3360 gaagccttgg tgatatacat ggagagtctg gccttagcac atgcagatga gaagtcctta 3420 ggtacaattc aacagtgttg tgatgccatt gatcacctat gccgcatcat tgaaaagaag 3480 catgtttctt taaataaagc aaagaaacga cgtttgccgc gaggatttcc accttccgca 3540 tcattgtgtt tattggatct ggtcaagtgg cttttagctc attgtgggag gccccagaca 3600 gaatgtcgac acaaatccat tgaactcttt tataaattcg ttcctttatt gccaggcaac 3660 agatccccta atttgtggct gaaagatgtt ctcaaggaag aaggtgtctc ttttctcatc 3720 aacacctttg aggggggtgg ctgtggccag ccctcgggca tcctggccca gcccaccctc 3780 ttgtaccttc gggggccatt cagcctgcag gccacgctat gctggctgga cctgctcctg 3840 gccgcgttgg agtgctacaa cacgttcatt ggcgagagaa ctgtaggagc gctccaggtc 3900 ctaggtactg aagcccagtc ttcacttttg aaagcagtgg ctttcttctt agaaagcatt 3960 gccatgcatg acattatagc agcagaaaag tgctttggca ctggggcagc aggtaacaga 4020 acaagcccac aagagggaga aaggtacaac tacagcaaat gcaccgttgt ggtccggatt 4080 atggagttta ccacgactct gctaaacacc tccccggaag gatggaagct cctgaagaag 4140 gacttgtgta atacacacct gatgagagtc ctggtgcaga cgctgtgtga gcccgcaagc 4200 ataggtttca acatcggaga cgtccaggtt atggctcatc ttcctgatgt ttgtgtgaat 4260 ctgatgaaag ctctaaagat gtccccatac aaagatatcc tagagaccca tctgagagag 4320 aaaataacag cacagagcat tgaggagctt tgtgccgtca acttgtatgg ccctgacgcg 4380 caagtggaca ggagcaggct ggctgctgtt gtgtctgcct gtaaacagct tcacagagct 4440 gggcttctgc ataatatatt accgtctcag tccacagatt tgcatcattc tgttggcaca 4500 gaacttcttt ccctggttta taaaggcatt gcccctggag atgagagaca gtgtctgcct 4560 tctctagacc tcagttgtaa gcagctggcc agcggacttc tggagttagc ctttgctttt 4620 ggaggactgt gtgagcgcct tgtgagtctt ctcctgaacc cagcggtgct gtccacggcg 4680 tccttgggca gctcacaggg cagcgtcatc cacttctccc atggggagta tttctatagc 4740 ttgttctcag aaacgatcaa cacggaatta ttgaaaaatc tggatcttgc tgtattggag 4800 ctcatgcagt cttcagtgga taataccaaa atggtgagtg ccgttttgaa cggcatgtta 4860 gaccagagct tcagggagcg agcaaaccag aaacaccaag gactgaaact tgcgactaca 4920 attctgcaac actggaagaa gtgtgattca tggtgggcca aagattcccc tctcgaaact 4980 aaaatggcag tgctggcctt actggcaaaa attttacaga ttgattcatc tgtatctttt 5040 aatacaagtc atggttcatt ccctgaagtc tttacaacat atattagtct acttgctgac 5100 acaaagctgg atctacattt aaagggccaa gctgtcactc ttcttccatt cttcaccagc 5160 ctcactggag gcagtctgga ggaacttaga cgtgttctgg agcagctcat cgttgctcac 5220 ttccccatgc agtccaggga atttcctcca ggaactccgc ggttcaataa ttatgtggac 5280 tgcatgaaaa agtttctaga tgcattggaa ttatctcaaa gccctatgtt gttggaattg 5340 atgacagaag ttctttgtcg ggaacagcag catgtcatgg aagaattatt tcaatccagt 5400 ttcaggagga ttgccagaag gggttcatgt gtcacacaag taggccttct ggaaagcgtg 5460 tatgaaatgt tcaggaagga tgacccccgc ctaagtttca cacgccagtc ctttgtggac 5520 cgctccctcc tcactctgct gtggcactgt agcctggatg ctttgagaga attcttcagc 5580 acaattgtgg tggatgccat tgatgtgttg aagtccaggt ttacaaagct aaatgaatct 5640 acctttgata ctcaaatcac caagaagatg ggctactata agattctaga cgtgatgtat 5700 tctcgccttc ccaaagatga tgttcatgct aaggaatcaa aaattaatca agttttccat 5760 ggctcgtgta ttacagaagg aaatgaactt acaaagacat tgattaaatt gtgctacgat 5820 gcatttacag agaacatggc aggagagaat cagctgctgg agaggagaag actttaccat 5880 tgtgcagcat acaactgcgc catatctgtc atctgctgtg tcttcaatga gttaaaattt 5940 taccaaggtt ttctgtttag tgaaaaacca gaaaagaact tgcttatttt tgaaaatctg 6000 atcgacctga agcgccgcta taattttcct gtagaagttg aggttcctat ggaaagaaag 6060 aaaaagtaca ttgaaattag gaaagaagcc agagaagcag caaatgggga ttcagatggt 6120 ccttcctata tgtcttccct gtcatatttg gcagacagta ccctgagtga ggaaatgagt 6180 caatttgatt tctcaaccgg agttcagagc tattcataca gctcccaaga ccctagacct 6240 gccactggtc gttttcggag acgggagcag cgggacccca cggtgcatga tgatgtgctg 6300 gagctggaga tggacgagct caatcggcat gagtgcatgg cgcccctgac ggccctggtc 6360 aagcacatgc acagaagcct gggcccgcct caaggagaag aggattcagt gccaagagat 6420 cttccttctt ggatgaaatt cctccatggc aaactgggaa atccaatagt accattaaat 6480 atccgtctct tcttagccaa gcttgttatt aatacagaag aggtctttcg cccttacgcg 6540 aagcactggc ttagcccctt gctgcagctg gctgcttctg aaaacaatgg aggagaagga 6600 attcactaca tggtggttga gatagtggcc actattcttt catggacagg cttggccact 6660 ccaacagggg tccctaaaga tgaagtgtta gcaaatcgat tgcttaattt cctaatgaaa 6720 catgtctttc atccaaaaag agctgtgttt agacacaacc ttgaaattat aaagaccctt 6780 gtcgagtgct ggaaggattg tttatccatc ccttataggt taatatttga aaagttttcc 6840 ggtaaagatc ctaattctaa agacaactca gtagggattc aattgctagg catcgtgatg 6900 gccaatgacc tgcctcccta tgacccacag tgtggcatcc agagtagcga atacttccag 6960 gctttggtga ataatatgtc ctttgtaaga tataaagaag tgtatgccgc tgcagcagaa 7020 gttctaggac ttatacttcg atatgttatg gagagaaaaa acatactgga ggagtctctg 7080 tgtgaactgg ttgcgaaaca attgaagcaa catcagaata ctatggagga caagtttatt 7140 gtgtgcttga acaaagtgac caagagcttc cctcctcttg cagacaggtt catgaatgct 7200 gtgttctttc tgctgccaaa atttcatgga gtgttgaaaa cactctgtct ggaggtggta 7260 ctttgtcgtg tggagggaat gacagagctg tacttccagt taaagagcaa ggacttcgtt 7320 caagtcatga gacatagaga tgatgaaaga caaaaagtat gtttggacat aatttataag 7380 atgatgccaa agttaaaacc agtagaactc cgagaacttc tgaaccccgt tgtggaattc 7440 gtttcccatc cttctacaac atgtagggaa caaatgtata atattctcat gtggattcat 7500 gataattaca gagatccaga aagtgagaca gataatgact cccaggaaat atttaagttg 7560 gcaaaagatg tgctgattca aggattgatc gatgagaacc ctggacttca attaattatt 7620 cgaaatttct ggagccatga aactaggtta ccttcaaata ccttggaccg gttgctggca 7680 ctaaattcct tatattctcc taagatagaa gtgcactttt taagtttagc aacaaatttt 7740 ctgctcgaaa tgaccagcat gagcccagat tatccaaacc ccatgttcga gcatcctctg 7800 tcagaatgcg aatttcagga atataccatt gattctgatt ggcgtttccg aagtactgtt 7860 ctcactccga tgtttgtgga gacccaggcc tcccagggca ctctccagac ccgtacccag 7920 gaagggtccc tctcagctcg ctggccagtg gcagggcaga taagggccac ccagcagcag 7980 catgacttca cactgacaca gactgcagat ggaagaagct catttgattg gctgaccggg 8040 agcagcactg acccgctggt cgaccacacc agtccctcat ctgactcctt gctgtttgcc 8100 cacaagagga gtgaaaggtt acagagagca cccttgaagt cagtggggcc tgattttggg 8160 aaaaaaaggc tgggccttcc aggggacgag gtggataaca aagtgaaagg tgcggccggc 8220 cggacggacc tactacgact gcgcagacgg tttatgaggg accaggagaa gctcagtttg 8280 atgtatgcca gaaaaggcgt tgctgagcaa aaacgagaga aggaaatcaa gagtgagtta 8340 aaaatgaagc aggatgccca ggtcgttctg tacagaagct accggcacgg agaccttcct 8400 gacattcaga tcaagcacag cagcctcatc accccgttac aggccgtggc ccagagggac 8460 ccaataattg caaaacagct ctttagcagc ttgttttctg gaattttgaa agagatggat 8520 aaatttaaga cactgtctga aaaaaacaac atcactcaaa agttgcttca agacttcaat 8580 cgttttctta ataccacctt ctctttcttt ccaccctttg tctcttgtat tcaggacatt 8640 agctgtcagc acgcagccct gctgagcctc gacccagcgg ctgttagcgc tggttgcctg 8700 gccagcctac agcagcccgt gggcatccgc ctgctagagg aggctctgct ccgcctgctg 8760 cctgctgagc tgcctgccaa gcgagtccgt gggaaggccc gcctccctcc tgatgtcctc 8820 agatgggtgg agcttgctaa gctgtataga tcaattggag aatacgacgt cctccgtggg 8880 atttttacca gtgagatagg aacaaagcaa atcactcaga gtgcattatt agcagaagcc 8940 agaagtgatt attctgaagc tgctaagcag tatgatgagg ctctcaataa acaagactgg 9000 gtagatggtg agcccacaga agccgagaag gatttttggg aacttgcatc ccttgactgt 9060 tacaaccacc ttgctgagtg gaaatcactt gaatactgtt ctacagccag tatagacagt 9120 gagaaccccc cagacctaaa taaaatctgg agtgaaccat tttatcagga aacatatcta 9180 ccttacatga tccgcagcaa gctgaagctg ctgctccagg gagaggctga ccagtccctg 9240 ctgacattta ttgacaaagc tatgcacggg gagctccaga aggcgattct agagcttcat 9300 tacagtcaag agctgagtct gctttacctc ctgcaagatg atgttgacag agccaaatat 9360 tacattcaaa atggcattca gagttttatg cagaattatt ctagtattga tgtcctctta 9420 caccaaagta gactcaccaa attgcagtct gtacaggctt taacagaaat tcaggagttc 9480 atcagcttta taagcaaaca aggcaattta tcatctcaag ttccccttaa gagacttctg 9540 aacacctgga caaacagata tccagatgct aaaatggacc caatgaacat ctgggatgac 9600 atcatcacaa atcgatgttt ctttctcagc aaaatagagg agaagcttac ccctcttcca 9660 gaagataata gtatgaatgt ggatcaagat ggagacccca gtgacaggat ggaagtgcaa 9720 gagcaggaag aagatatcag ctccctgatc aggagttgca agttttccat gaaaatgaag 9780 atgatagaca gtgcccggaa gcagaacaat ttctcacttg ctatgaaact actgaaggag 9840 ctgcataaag agtcaaaaac cagagacgat tggctggtga gctgggtgca gagctactgc 9900 cgcctgagcc actgccggag ccggtcccag ggctgctctg agcaggtgct cactgtgctg 9960 aaaacagtct ctttgttgga tgagaacaac gtgtcaagct acttaagcaa aaatattctg 10020 gctttccgtg accagaacat tctcttgggt acaacttaca ggatcatagc gaatgctctc 10080 agcagtgagc cagcctgcct tgctgaaatc gaggaggaca aggctagaag aatcttagag 10140 ctttctggat ccagttcaga ggattcagag aaggtgatcg cgggtctgta ccagagagca 10200 ttccagcacc tctctgaggc tgtgcaggcg gctgaggagg aggcccagcc tccctcctgg 10260 agctgtgggc ctgcagctgg ggtgattgat gcttacatga cgctggcaga tttctgtgac 10320 caacagctgc gcaaggagga agagaatgca tcagttattg attctgcaga actgcaggcg 10380 tatccagcac ttgtggtgga gaaaatgttg aaagctttaa aattaaattc caatgaagcc 10440 agattgaagt ttcctagatt acttcagatt atagaacggt atccagagga gactttgagc 10500 ctcatgacaa aagagatctc ttccgttccc tgctggcagt tcatcagctg gatcagccac 10560 atggtggcct tactggacaa agaccaagcc gttgctgttc agcactctgt ggaagaaatc 10620 actgataact acccgcaggc tattgtttat cccttcatca taagcagcga aagctattcc 10680 ttcaaggata cttctactgg tcataagaat aaggagtttg tggcaaggat taaaagtaag 10740 ttggatcaag gaggagtgat tcaagatttt attaatgcct tagatcagct ctctaatcct 10800 gaactgctct ttaaggattg gagcaatgat gtaagagctg aactagcaaa aacccctgta 10860 aataaaaaaa acattgaaaa aatgtatgaa agaatgtatg cagccttggg tgacccaaag 10920 gctccaggcc tgggggcctt tagaaggaag tttattcaga cttttggaaa agaatttgat 10980 aaacattttg ggaaaggagg ttctaaacta ctgagaatga agctcagtga cttcaacgac 11040 attaccaaca tgctactttt aaaaatgaac aaagactcaa agccccctgg gaatctgaaa 11100 gaatgttcac cctggatgag cgacttcaaa gtggagttcc tgagaaatga gctggagatt 11160 cccggtcagt atgacggtag gggaaagcca ttgccagagt accacgtgcg aatcgccggg 11220 tttgatgagc gggtgacagt catggcgtct ctgcgaaggc ccaagcgcat catcatccgt 11280 ggccatgacg agagggaaca ccctttcctg gtgaagggtg gcgaggacct gcggcaggac 11340 cagcgcgtgg agcagctctt ccaggtcatg aatgggatcc tggcccaaga ctccgcctgc 11400 agccagaggg ccctgcagct gaggacctat agcgttgtgc ccatgacctc caggttagga 11460 ttaattgagt ggcttgaaaa tactgttacc ttgaaggacc ttcttttgaa caccatgtcc 11520 caagaggaga aggcggctta cctgagtgat cccagggcac cgccgtgtga atataaagat 11580 tggctgacaa aaatgtcagg aaaacatgat gttggagctt acatgctaat gtataagggc 11640 gctaatcgta ctgaaacagt cacgtctttt agaaaacgag aaagtaaagt gcctgctgat 11700 ctcttaaagc gggccttcgt gaggatgagt acaagccctg aggctttcct ggcgctccgc 11760 tcccacttcg ccagctctca cgctctgata tgcatcagcc actggatcct cgggattgga 11820 gacagacatc tgaacaactt tatggtggcc atggagactg gcggcgtgat cgggatcgac 11880 tttgggcatg cgtttggatc cgctacacag tttctgccag tccctgagtt gatgcctttt 11940 cggctaactc gccagtttat caatctgatg ttaccaatga aagaaacggg ccttatgtac 12000 agcatcatgg tacacgcact ccgggccttc cgctcagacc ctggcctgct caccaacacc 12060 atggatgtgt ttgtcaagga gccctccttt gattggaaaa attttgaaca gaaaatgctg 12120 aaaaaaggag ggtcatggat tcaagaaata aatgttgctg aaaaaaattg gtacccccga 12180 cagaaaatat gttacgctaa gagaaagtta gcaggtgcca atccagcagt cattacttgt 12240 gatgagctac tcctgggtca tgagaaggcc cctgccttca gagactatgt ggctgtggca 12300 cgaggaagca aagatcacaa cattcgtgcc caagaaccag agagtgggct ttcagaagag 12360 actcaagtga agtgcctgat ggaccaggca acagacccca acatccttgg cagaacctgg 12420 gaaggatggg agccctggat gtgaggtctg tgggagtctg cagatagaaa gcattacatt 12480 gtttaaagaa tctactatac tttggttggc agcattccat gagctgattt tcctgaaaca 12540 ctaaagagaa atgtcttttg tgctacagtt tcgtagcatg agtttaaatc aagattatga 12600 tgagtaaatg tgtatgggtt aaatcaaaga taaggttata gtaacatcaa agattaggtg 12660 aggtttatag aaagatagat atccaggctt accaaagtat taagtcaaga atataatatg 12720 tgatcagctt tcaaagcatt tacaagtgct gcaagttagt gaaacagctg tctccgtaaa 12780 tggaggaaat gtggggaagc cttggaatgc ccttctggtt ctggcacatt ggaaagcaca 12840 ctcagaaggc ttcatcacca agattttggg agagtaaagc taagtatagt tgatgtaaca 12900 ttgtagaagc agcataggaa caataagaac aataggtaaa gctataatta tggcttatat 12960 ttagaaatga ctgcatttga tattttagga tatttttcta ggttttttcc tttcatttta 13020 ttctcttcta gttttgacat tttatgatag atttgctctc tagaaggaaa cgtctttatt 13080 taggagggca aaaattttgg tcatagcatt cacttttgct attccaatct acaactggaa 13140 gatacataaa agtgctttgc attgaatttg ggataacttc aaaaatccca tggttgttgt 13200 tagggatagt actaagcatt tcagttccag gagaataaaa gaaattccta tttgaaatga 13260 attcctcatt tggaggaaaa aaagcatgca ttctagcaca acaagatgaa attatggaat 13320 acaaaagtgg ctccttccca tgtgcagtcc ctgtcccccc ccgccagtcc tccacaccca 13380 aactgtttct gattggcttt tagctttttg ttgttttttt ttttccttct aacacttgta 13440 tttggaggct cttctgtgat tttgagaagt atactcttga gtgtttaata aagttttttt 13500 ccaaaagta 13509 <210> 7 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> DNA-PKcs sense primer <400> 7 gaattcaagc gagtccgtgg gaaggcc 27 <210> 8 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> DNA-PKcs anti-sense primer <400> 8 gtcgacttcg ctgcttatga tgaaggg 27 <210> 9 <211> 424 <212> DNA <213> human AMPK gamma1 <400> 9 catacgatgt tccagattac gctagcttgg gtggtcatat ggccatggag gccccgggga 60 tccgaattcg cggccgcgtc gaccgcggcc gcgtcgacca ggactccttt aaaccgcttg 120 tctgcatttc tcctaatgcc agcttgtttg atgctgtctc ttcattaatt cggaacaaga 180 tccacaggct gccagttatt gacccagaat caggcaatac tttgtacatc ctcacccaca 240 agcgcattct gaagttcctc aaattgttta tcactgagtt ccccaagcca gagttcatgt 300 ccaagtctct ggaagagcta cagattggca cctatgccaa tattgctatg gttcgcacta 360 ccacccccgt ctatgtggct ctggggattt ttgtacagca tcgagtctca gccctgccag 420 tggt 424  

Claims (6)

1) treating AMPKγ1, its binding partner DNA-PKcs and candidates that inhibit the mutual binding between the AMPKγ1 and DNA-PKcs (experimental group); 2) measuring the mutual binding activity (positive control group) between AMPKγ1 and DNA-PKcs and the mutual binding activity of the experimental group of step 1), respectively; And, 3) A method for screening a drug for inhibiting proliferation and / or metastasis of cancer, comprising selecting a candidate substance that inhibits the mutual binding activity than a positive control group. 1) treating AMPKγ1, its binding partner DNA-PKcs and candidates that inhibit the mutual binding between the AMPKγ1 and DNA-PKcs (experimental group); 2) measuring the mutual binding activity (positive control group) between AMPKγ1 and DNA-PKcs and the mutual binding activity of the experimental group of step 1), respectively; And, 3) A method for screening a neuroprotective drug comprising the step of selecting a candidate to inhibit the cross-linking activity than a positive control group. The method of claim 1 or 2, wherein the AMPKγ1 and DNA-PKcs of step 1) are described as SEQ ID NO: 1 and SEQ ID NO: 2, respectively. The method according to claim 1 or 2, characterized in that phosphorylation of AMPKγ1 is inhibited by inhibition of cross-linking between DNA-PKcs and AMPKγ1. The method of claim 1 or 2, wherein step 1) the candidate is a natural compound, synthetic compound, RNA, siRNA, DNA, polypeptide, enzyme, protein, ligand, antibody, antigen, bacterial or fungal metabolite and bioactive molecule. Method selected from the group consisting of. The method of claim 1 or 2, wherein the measurement of the interaction activity of step 2) is performed after binding the remaining protein having a fluorescent substance attached to a protein chip in which one of AMPKγ1 and DNA-PKcs is immobilized on a solid substrate. Method for measuring the detector, a protein attached to a solid substrate, the remaining protein and the remaining protein specifically bound, the method of measuring the detector after binding the antibody labeled with the detector, without the label of the detector And a surface plasmon resonance (SPR) method for measuring plasmon resonance changes on a surface in real time, and a surface plasmon resonance imaging (SPRI) method for imaging and confirming an SPR system.

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