CN1681939A - Inducible focal adhesion kinase cell assay - Google Patents

Abstract

The cell-based assay of the present invention exploits the biology of FAK in conjunction an inducible gene expression system to exogenously control FAK expression and FAK phosphorylation at the tyrosine residue at position 397 (Y<397>). The cell-based assay of the present invention is flexible and can measure FAK phosphorylation at Y397, total FAK phosphorylation, identify mutant FAK proteins and measure a combination of protein and phosphotyrosine.

Description

Derivable focal adhesion kinase test cell line

Background of invention

The present invention relates to be used to induce the method and composition of focal adhesion kinase (FAK) genetic expression, this focal adhesion kinase (FAK) genes encoding participation somatomedin is replied the signal protein with cell migration, and and disease-related.The invention still further relates to the evaluation of FAK inhibitor.

FAK is a kind of kytoplasm nonreceptor tyrosine kinase.The FAK transduction is controlled the various kinds of cell path and the process that comprise cell proliferation, migration, form and cell survival from the signal of different types of stimulation (as integral protein, cytokine, chemokine and somatomedin).Except most types of organizations express, found FAK at most of human cancers, especially level raises in the height infectivity shifts.The expression of the relevant nonkinase (FRNK) of dominant negative (dominant-negative) FAK causes cell circular in reference's tumour cell, irreversible forfeiture of adhesion plaque and necrocytosis subsequently.In addition, the controlled expression of FRNK reduces the tyrosine phosphorylation of FAK, illustrates that the inhibition of FAK phosphorylation can produce the therapeutic index of treatment human cancer.

Though cause FAK activatory precise mechanism not fully aware of, but think that causing the FAK enzymic activity of Y397 (being arranged in the tyrosine residues in 397 sites) phosphorylation is committed step (Guan at the integral protein signal transduction, JL, Int.J.Biochem.Cell.Biol.29:1085-96,1997).For extracellular matrix (ECM) albumen is connected with nucleus with the cell actin cytoskeleton, thereby regulate the genetic expression of cellular form, weave construction and adhesion mediation, it is very important striding film integral protein acceptor.Integral protein acceptor and FAK are located on the adhesion plaque site and will cause FAK in Y397 residue phosphorylation, and the SH2 that produces a Src family Tyrosylprotein kinase stops the site.Src causes FAK at tyrosine residues place, different downstream with the combination of Tyrosine O-phosphate FAKY397, comprises that Y576, Y577, Y861 and Y925 are preferentially by phosphorylation.The phosphorylation of FAK tyrosine residues (Y576/Y577) makes the FAK kinase activity strengthen, and transduction signal is regulated cytoskeleton reorganization, cell proliferation, cell survival and cell migration.

Owing to participate in the kinases of integral protein signal cascade and the diversity of substrate, need the design test special to specific kinases.The objective of the invention is design and develop a kind of FAK drug discovery approach, the biological chemistry mechanism of this road tracking FAK.Some exogenous stimulations can both cause the FAK phosphorylation, as 1) integral protein combines (for example, integral protein β 1 combines with fibronectin) with the ECM part; 2) cytokine or chemokine stimulate (for example, endothelin 1/2, bombesin or PMA); 3) factors stimulated growth of tyrosine kinase receptor (for example, PDGFBB); With 4) alphab-integrin antibodies crosslinked (for example, anti--β 1).On the contrary, making the most feasible exogenous control of FAK inactivation is exactly contact (for example, the cell suspension) of blocking cell-cell and cell-ECM.

Summary of the invention

The present invention relates to identify the method for cell-activity (cell-active) inhibitor of focal adhesion kinase (FAK), comprising:

(a) add the genetic expression that inductor is induced coding FAK in mammalian cell, wherein said mammalian cell is by described stable gene transfection, and described gene is expressed when described inductor exists;

(b) add test compound;

(c) utilize FAK trapping agent (capture agent) to catch the FAK that expresses; And

(d) phosphorylation of the described FAK of detection.

Embodiments of the present invention provide a kind of Cytotoxic method of measuring test compound, comprising: with the stable gene transfection mammalian cell of coding FAK, wherein said gene is expressed when inductor exists; Add inductor and induce the genetic expression of described coding FAK; Add test compound; Cytotoxicity is added to described cell; And, detect the cytotoxicity of described test compound.In some embodiments, the colorimetric by cytotoxicity is converted and determines the cytotoxicity of test compound, and wherein the inversion quantity of cytotoxicity is directly proportional with viable count.

Embodiments of the present invention provide a kind of mammalian cell with the nucleic acid molecule stable transfection of recombinating, the nucleic acid molecule of wherein said reorganization is selected from SEQ ID NO:5, SEQ ID NO:6, SEQID NO:7 and SEQ ID NO:8, and the expression of wherein said sequence needs the existence of inductor.

Embodiments of the present invention provide a kind of mammalian cell with the nucleic acid molecule stable transfection of recombinating, the nucleic acid molecule encoding of this reorganization contains the albumen that is selected from SEQ ID NO:1,2,3 and 4 sequence, and wherein said proteic expression needs the existence of inductor.

Description of drawings

Fig. 1 has shown and has utilized horseradish peroxidase-link coupled phosphotyrosine antibody (pY54 ^HRP) detect the synoptic diagram of the FAK of phosphorylation.

Fig. 2 shown and utilized not link coupled phosphotyrosine antibody (pY54), uses the second antibody of the horseradish peroxidase of mouse to detect the synoptic diagram of the FAK of phosphorylation afterwards.

Fig. 3 has shown the synoptic diagram of FAK inducibility of the present invention based on the test of cell.

Detailed Description Of The Invention

The present invention relates to the FAK inducibility take the test of cell as the basis. Described test take cell as the basis has utilized biology and the inducible gene expression system of FAK, thus outer seedbed control FAK expression and in the FAK phosphorylation of 397 site tyrosine residues (Y397). By utilizing FAK^Y397Phosphorylation-specific is take the test of cell as the basis, rather than common phosphotyrosine system, can avoid identifying the false positive inhibitor. The test of the present invention take cell as the basis is flexibly, and it can measure the FAK phosphorylation of Y397 position, whole FAK phosphorylation, identifies that the FAK albumen of sudden change is also measured albumen and the combination of phosphotyrosine.

The test of FAK inducibility of the present invention take cell as the basis has advantage, because it has strictly controlled the expression of the dystopy foundation level of FAK, and makes FAK gene expression disinthibite rapidly (de-repression) by exogenous stimulation. Test take cell as the basis is flexibly, like this such as phosphotyrosine FAK^Y397, whole mensuration of some combinations of FAK phosphotyrosine distribution maps, FAK or mutain or albumen and phosphotyrosine, final reading is mechanical relevant with the biology of FAK. In addition, some FAK inhibitor the present invention have been successfully used to identify. As used herein, " strict control " refers to the controlled expression of the FAK gene of appearance when the external source sexual stimulus exists. In other words, the invention provides a kind of FAK of inducing gene expression system, in this system, under suitable derivant existence condition, can induce FAK to express. The invention provides the method that a kind of FAK of inducing expresses, wherein regulate expression FAK gene and the viability of cell is not had a negative impact.

Embodiments of the present invention relate to a kind of FAK of screening inhibitor take cell as the basis test. Test take cell as the basis has utilized biology and the inducible gene expression system of FAK, thereby exogenous control FAK expresses and the FAK phosphorylation of 397 place's tyrosine residues (Y397) in the site. Described mechanically relevant with the biology of FAK take cell as the test on basis, and the variation in the mensuration FAK phosphorylation. By using FAK^Y397Phosphorylation-specific is take the test of cell as the basis, rather than common phosphotyrosine system, can avoid identifying the false positive inhibitor. Test take cell as the basis of the present invention is flexibly, and it can measure FAK phosphorylation, whole FAK phosphorylation, identifies that the FAK albumen of sudden change is also measured albumen and the combination of phosphotyrosine.

Embodiments of the present invention provide the method for cell-activity inhibitor of a kind of FAK of evaluation, comprising: with the stable gene transfection mammalian cell of coding FAK, wherein said gene is expressed when derivant exists; Add derivant and induce the gene expression of described coding FAK; Add test compound; Utilize the FAK agent for capturing to catch the FAK that expresses; Make the FAK of seizure be exposed to the antibody of anti--phosphotyrosine; And detect the phosphorylation of described FAK. In some embodiments, the degree of described FAK phosphorylation determines by anti--phosphoric acid-tyrosine antibody and the combination of the FAK that catches, and wherein the phosphorylation amount of the resisting of the FAK that catches of combination-phosphoric acid-tyrosine antibody amount and described FAK is proportional.

In some embodiments of the present invention, the method for identifying cell-activity inhibitor of FAK comprises: mammalian cell is coated on optional step on the first solid phase. Described the first solid phase is the hole of the first microtiter plate preferably. In yet another embodiment of the present invention, before the FAK that catch to express, will be coated on lysis on the first solid phase with lysis buffer. The optional solubility detergent that comprises of described lysis buffer. In some embodiments, the FAK agent for capturing is coated on the second solid phase, and this second solid phase is the hole of the second microtiter plate preferably.

In some embodiments, test compound contains the FAK phosphorylation that suppresses the Y397 place.

Embodiments of the present invention provide a kind of mensuration test compound Cytotoxic method, comprising: with the stable gene transfection mammalian cell of coding FAK, wherein said gene is expressed when derivant exists; Add derivant and induce the expression of the gene of described coding FAK; Add test compound; Cytotoxicity is added to described cell; And, the cytotoxicity of detection test compound. In some embodiments, the colorimetric by cytotoxicity is converted and determines the cytotoxicity of test compound, and wherein the inversion quantity of cytotoxicity and viable count are proportional.

In some embodiments of the present invention, measuring the Cytotoxic method of test compound comprises: mammalian cell is coated on optional step on the solid phase. This solid phase is the hole of the first microtiter plate preferably.

Embodiments of the present invention provide the method for the cell-activity inhibitor of a kind of evaluation focal adhesion kinase (FAK), comprise: bag is by the mammalian cell of a group homogeneous on first solid phase, the cell and first solid phase are adhered to, the be encoded stable gene transfection of FAK of wherein said cell, and wherein said gene is expressed when inductor exists; Add inductor and induce the genetic expression of described coding FAK; Add test compound; Adherent cell is dissolved discharge cell lysate; By second solid phase, the described FAK trapping agent and second solid phase are adhered to FAK trapping agent bag; The FAK trapping agent that cell lysate is exposed to adhere to makes the FAK trapping agent catch FAK; Make the FAK of seizure be exposed to the antibody of anti--phosphoric acid-tyrosine; And, measure anti--phosphoric acid-tyrosine antibody and combine with the FAK's that is caught, wherein resist with the FAK bonded of being caught-amount of phosphoric acid-tyrosine antibody amount and described FAK phosphorylation is proportional.

Another embodiment of the invention provides the mammalian cell with the nucleic acid molecule stable transfection of reorganization, the nucleic acid molecule of wherein said reorganization is selected from SEQ ID No:5, SEQ ID No:6, SEQ IDNo:7 and SEQ ID No:8, and the expression of wherein said sequence needs the existence of inductor.

Embodiments of the present invention provide the mammalian cell with the nucleic acid molecule stable transfection of reorganization, and the nucleic acid molecule encoding of this reorganization comprises and is selected from SEQ ID No:1, the albumen of 2,3 and 4 sequence, and wherein said proteic expression needs the existence of inductor.

FAK is also known to be albumen-Tyrosylprotein kinase 2, PTK2.Any active FAK variant can both use in said determination.Also can in mensuration, use the non-activity mutant to do various contrasts.Can in said determination, use other FAK variants, comprise: have 1052 amino acid (SEQ ID NO:1) at 153012 wild-type (WT) human FAK; FAK engages variant, as Andre, and E.﹠amp; Becker-Andre, M. the expression of N-terminal brachymemma shape human focal adhesion kinases in the brain, Biochem.Biophys.Res.Commun.190:140-147,1993 (having described 879 amino acid whose variants of AAA35819,431 amino acid whose variants of PC1226 and 554 amino acid whose variants of PC1227) describe; 570 catalytic domains of the FAK of XP_050337; The mouse FAK of NP_032008.1, it and human FAK have 1023/1053 amino acid whose identity (97%); The variant of the mouse FAK carboxyl brachymemma of AAH30180.1, the 1-903 amino acid of it and human FAK has 878/904 amino acid whose identity (97%); The rat FAK of NP_037213.1, it and human FAK have 1020/1055 amino acid whose identity (96%); The FAK variant of JC5494, it and human FAK have 1017/1055 amino acid whose identity (96%); The chicken FAK variant of Q00944, it and human FAK have 988/1054 amino acid whose identity (93%); The chicken FAK variant of A45388, it and human FAK have 965/1029 amino acid whose identity (93%); Synthetic FAK mutant, comprise FAK Y397F (SEQ ID NO:2), K454R (SEQ ID NO:3), FRNK (the N-terminal truncate that the FAK residue 694-1052 of promotor MET is arranged before containing) (SEQ ID NO:4), comprise FAK Y397D with various phosphorylation stand-in, Y397E, Y577D, Y577E, Y861D, Y861E, Y925D, Y925E and combination thereof; CD2-FAK syzygy (the activated FAK of structure and the syzygy of CD2) is as J Biol.Chem. (1994) such as Chan P; 269 (32): 20567-74 is described.

In this article, term " inductor " is medium (agent), compound or the chemical of at least 6 times of signal to noise ratios of a kind of generation.The example of inductor includes but not limited to: Mifepristone (Mifepristone) (Ru486) and other anti-Progesterone, as Org31806 and Org31376.Referring to O ' Malley etc., Cell, 69,703-713. (1992).In this article, the term trapping agent is a kind of medium, compound or the chemical that can catch any form focal adhesion kinase, and comprising with histidine residues, streptavidin or other has the mark of similar affinity to come the FAK of mark.Described trapping agent includes but not limited to Tyrosine O-phosphate FAK ^Y397Specific antibody, common phosphotyrosine antibody, anti-FAK antibody, anti-histidine mark antibody and being beneficial to captures the molecule that contains vitamin H of the FAK that avidin modifies.In some embodiments of the present invention, trapping agent comprises the combination of one or more antibody, includes but not limited to goat anti-rabbit antibodies and Tyrosine O-phosphate FAK ^Y397The combination of specific antibody.

In this article, term " anti-phosphoric acid-tyrosine antibody " includes but not limited to Tyrosine O-phosphate FAK ^Y397Specific antibody and common phosphotyrosine antibody, wherein the latter can discern the tyrosine residues of any phosphorylation, includes but not limited to the tyrosine residues in FAK site 397.

In this article, cytotoxicity is reagent, chemical or the compound that is used to estimate cell survival.The example of cytotoxicity includes but not limited to especially be fit to the tetrazolium (for example MTT, XTT, WST-1) of this kind analysis.

MTT is a kind of yellow tetrazolium, and it is cut into purple Jia Za (fornazan) crystal by the metabolic activity cell.Utilize ELISA reader or other spectrophotometer equipment can use spectrophotometric standard measure dissolved first Za product.Survey as absorbancy, the live body amount is directly related with the purple Jia Za crystal amount of formation.

Utilize inducibility FAK gene expression system to implement embodiments of the present invention, this system has the ability that the tight regulatory gene of making peace is expressed.In embodiments of the present invention, provide a kind of inducibility FAK gene expression system, it comprises that regulating transgenosis is the system that FAK expresses.When inductor did not exist, FAK expressed " closing ", and when inductor existed, FAK expressed " unlatching ".This system is by two genomic constitutions: one of them coding and regulating albumen, another interested transgenosis of inducing of encoding.Expressing described adjusting albumen is handled by the promotor that makes up.Derivable FAK transgenosis contains promotor, and this promotor is by forming with the minimal promoter that can be connected in conjunction with the multiple copied of the proteic binding site of described adjusting.

In embodiments of the present invention, described adjusting albumen is transcription factor, it is formed by people's progesterone receptor ligand binding domain of yeast GAL4DNA land, brachymemma with from the people p65 region of activation of NF-κ B, is beneficial to and closely regulates described transgenosis in basal expression.The proteic plasmid of coding and regulating comprises the GAL4 promotor, and this promotor has formed regenerative feedback loop (100p) and has been beneficial to part processing generation reaction rapidly.Utilize the proteic expression of the exogenous regulating and controlling of small molecules part.Combine with the species selection of described genetically modified promotor by described adjusting albumen, and realize tight adjusting by the proteic ligand dependent conformational activation of described adjusting.

Therefore, in some embodiments, invention has the following advantages:

But ■ utilizes inducible system to induce FAK in cell, and the tight inhibition of gene expression of this system energy when not induced produces the viable cell clone.

The inhibitor of FAK kinase activity is identified in the detection of ■ phosphoric acid FAK.Some methods that detect phosphorylation FAK comprise: identify the FAK inhibitor based on the analysis of polyacrylamide gel electrophoresis with based on the analysis of ELISA.The detection system that also can use other to be fit to.

■ test can be used for detecting whole FAK albumen, all by the FAK albumen of phosphorylation, or in given tyrosine place (for example, tyrosine 397) by the FAK albumen of phosphorylation.

■ can be a screening FAK inhibitor in system's body on basis in order to cell, because transfected cell is a tumorigenicity, and can induce FAK in vivo by giving the feeding animal Mifepristone.Shown the intravital effectiveness of this system.

Comparative example

Comparative example 1

For hope improves test signal and noise, in different cell backgrounds, attempt producing FAK or the FAK mutain of stablizing ectopic expression.But, because these cell major parts have shown susceptibility to the variation of FAK protein level, and always can not form exploitation and clone based on the survival of the test of cell, these effort are proved to be unsuccessful.The cell of the endogenous FAK of expression from low to medium level, the FAK that tolerates no more than twice endogenous levels as NIH3T3 l cell or A2058 metastatic human melanoma cell expresses.Like this, owing to previously described similar reason: stimulate and reproducibility bad, high background noise and do not benefit drug discovery proves these clones and is not suitable for pilot development.Therefore find that in containing the cell of natural FAK the non-abduction delivering of FAK also is not suitable for the research of inducing FAK to express.

Comparative example 2

In order to develop a kind of middle flux and high flux screening (ELISA system) of both having helped, can follow the trail of the FAK test based on cell of FAK biological chemistry mechanism again, taked a large amount of measures to develop the FAK biology.For example, by at extracellular matrix protein (ECM) albumen such as fibronectin or collagen protein, or other ECM such as matrigel go up inoculating cell, and the adherent cell of attempting simulation FAK stimulates, but it is ineffective that this measure is proved to be, and makes FAK stimulate bad and determined by cell type fully.In addition, pass through pY54 ^HRP(horseradish peroxidase (HRP)-link coupled phosphotyrosine antibody) detection assay ECM-matrigel-inductive cell attachment can not produce the FAK phosphorylation to stimulate, and uses the anti-mouse of secondary separately ^HRP(2 ⁰M ^HRP) control test make the non-specific growth of the test signal in the hole, hatched in this hole and come comfortable matrigel to go up the lysate of stimulated cells.When using pY54 ^HRPWith 2 ⁰M ^HRPDuring combination, signal to noise ratio (S/N) from 1.0 to 1.7, and pY54 (not coupling phosphotyrosine antibody) and 2 ⁰M ^HRPThe signal to noise ratio of associating generation is 1.0-2.4.This explanation and can affirming, the non-specific growth of test signal are that the cross reactivity by some matrigel pollutent in the cell lysate causes.Therefore, above-mentioned exploitation is infeasible based on the measure of the FAK test of cell.

The result is when also showed cell is adhered to ECM matrigel or fibronectin, and the twice of faint (modest) and not too suitable (suboptimal) to triple FAK phosphorylation signal to noise ratio stimulates.The type of adhering to time, detection and capture antibodies by change cell quantity, cell-ECM, and utilize the combined stimulation of endotheliocyte element-1 and phorbol 12-tetradecanoic acid 13-acetate (PMA) that these test conditionss are further optimized, but the non-revulsion that shows these FAK stimulations is infeasible, can not reproduce and be not suitable for pilot development.

Comparative example 3

Because an integral protein bunch collection makes the FAK phosphorylation, utilizes the ﹠ beta 1 integrin specific antibody that the antibody linked feasibility of integral protein acceptor is studied.But because the indirect method that FAK stimulates causes high mutability and not reproducible, it is unsuccessful that these effort also prove.For example, because optimal stimulus needs temperature sensitivity (temperature-sensitive) step, this step elapsed time and be difficult to management, integral protein β 1 is crosslinked even all be very loaded down with trivial details for middle flux screening.In addition, because the ECM loose contact reduces background noise with cell fixation cell survival is descended on polystyrene plate, institute is so that these non-inductive expression systems are not suitable for pilot development.

Comparative example 4

In order to accelerate the pilot development of FAK based on cell, carried out producing the trial of stable clone, these clones can express the FAK albumen of wild-type or sudden change in various cell backgrounds.The clone who produces contains FAK cDNA transcript, this transcript code set moulding activated protein (CD2FAK), membrane-bound tyrosine-inefficacy (dead) albumen (CD2FAK ^Y397F), or lose the kytoplasm FAK mutant (FAK for example of crucial downstream tyrosine residues ^Y861F, FAK ^Y925FAnd FAK ^Y861F/Y922F).On the basis of FAK biology understanding, these biological tool are carried out diplomatic design, to solve problem described above and to develop strong (robust) and the reproducible test that is of value to drug development based on cell.

Even obtained the contrast clone of energy expression vector configuration or the proteic work of LacZ, attempted producing expression FAK ^WTStable clone also be unsuccessful.Design FAK mutant configuration cuts off downstream FAK phosphorylation and keeps Y397 position FAK phosphorylation and kinase activity (FAK ^Y861F, FAK ^Y925FAnd FAK ^Y861F/Y925F), or no longer need integral protein acceptor (CD2FAK ^WTAnd CD2FAK ^Y397F), the transfection that proves these FAK mutant configurations all is unsuccessful.Express low to medium level endogenous FAK such as the cell of metastatic human melanoma A2058 in identify the clone who lives.But A2058FAK ^WTThe clone shows that the FAK level has only increased about 2 times, and this illustrates that it is not suitable for FAK pilot development based on cell.

Except carrying out exogenous stimulation, control basic FAK phosphorylation, and attempt outside the ectopic expression FAK clone, but use the Tet-On/Tet-Off inducible system to control FAK with tsiklomitsin ^WTAnd tyrosine-inefficacy FAK ^Y397FCDNA.But do not detect FAK ^WTOr FAK ^Y397FTransfectant.Owing to fail to identify the clone that lives, just can not determine at FAK ^WTOr FAK ^Y397F" omitting (leakiness) " in the Tet-transfectant promptly regulated not enough level to basal expression.Tet-On ^TM/ Tet-Off ^TMSystem shows the variable regulation and control that target protein is expressed, so it is not suitable for the adjusting of lethal gene such as FAK.

Embodiment

With wild-type FAK ^WT(SEQ ID NO:5), tyrosine-inefficacy FAK ^Y397F(SEQ ID NO:6), kinases-inefficacy FAK ^K454R(SEQ ID NO:7) relevant nonkinase with dominant negative FAK (FRNK) (SEQ ID NO:8) inserts fragment as BarnHI-Apal or Kpnl-Apal, is cloned into GeneSwitch ^TMGo in the pGeneV5/His A-carrier framework.As used herein, term " FAK of genes encoding " comprises but is unlimited to SEQ ID NO:1-4.These configurations are based on open (the GenBank accession number L13616) of human FAK sequence, referring to Whitney, G.S. etc., DNA Cell Biol.12 (9), 823-830 (1993), and every kind of configuration is all definite through checking order, and with sequence alignment is disclosed.Transforming dna structure comprises unique and is different from the specificity 5 of prior art ' and 3 ' restriction site.In addition, the 3 ' end of also transforming some configuration comprises epi-position such as the V5 and the histidine mark of mark.

Select many clones to be used for transfection, it comprises NIH Swiss Mouse inoblast NIH3T3 (ATCC accession number CCL-92), people's epidermoid carcinoma A431 (ATCC accession number CRL1555) and people's glioblastoma astrocytoma U87MG (ATCC accession number HTB-14).Every kind of clone has shown that all it was applicable to the unique property of expressing FAK.For example, the NIH3T3 cell makes FAK specific specificity cross expression and be easy to transfected, the endogenous FAK of A431 cell expressing medium level also provides the tumour that more can represent FAK natural surroundings background, and the U87MG cell lacks the FAK inactivation conditioning agent PTEN (a kind of tumor suppression acid phosphatase (tumorsuppressor phosphatase)) of supposition.Utilize the Gene Jammer of Stratagene ^TMTransfection agents is regulated carrier and pGene with pSwitch ^VectorV5His, or pGeneFAK ^WTV5His, or pGeneFAK mutant (pGeneFAK ^K454RV5His, and pGeneFAK ^Y397FV5His) cotransfection is gone into the A431 cell.Similarly, transforming NIH3T3 and U87MG cell comes coexpression pSwitch to regulate albumen and interested specificity pGene configuration.Select Totomycin and Zeocin resistance clone, propagation (expand) is screened to analyze by western and RT-PCR in substratum.Many derivable clones are comprised: A431:FAK ^WTV5His, A431:FAK ^Y397FV5His, A431:FAK ^K454RV5His, A431: carrier, NIH3T3:FAK ^WTV5His, NIH3T3:FAK ^Y397FV5His, NIH3T3: carrier, U87MG:FAK ^WTV5His and U87MG:FAK ^Y397FV5His successfully separates, and selects, and determines and confirmation.

The FAK inducibility test determination of embodiment of the present invention the FAK phosphorylation of Y397 position.With A431:FAK ^WTV5His cell (about 1.0 * 10 ⁴-1.0 * 10 ⁷Individual cell) be seeded at the bottom of the U type on 96 orifice plates, before the incubation that spends the night with the 0.1nM Mifepristone, at 37 ℃, 5%CO ₂Adhered to 4-6 hour.Then, with A431:FAK ^WTThe not treated placement of V5His inductive cell or at 37 ℃, 5%CO ₂With suppressing compound treatment 30 minutes, and at RIPA lysis buffer (50mM Tris-HCl, pH7.4,1%NP-40,0.25% Sodium desoxycholate, 150mM NaCI, 1mM EDTA, 1mM Na ₃VO ₄, (pellet) Complete of 1mM NaF and every 50ml solution ^TMNo EDTA proteinase inhibitor particle) cracking in.The total protein (100 μ l) of about 45 μ g is transferred to goat antirabbit flat board with 0.35 μ g/ hole anti-FAK phosphoric acid specificity Y397 antibody sandwich, to catch and to detect subsequently the FAK albumen (see figure 3) of phosphorylation.

In the T25 flask, inoculation NIH3T3:FAK ^WTV5His clone and NIH3T3:FAK ^Y397FV5His is cloned into degree of converging and is about 80%, not treated placement or at 37 ℃, 5%CO ₂Stimulated about 16 hours with the 0.1nM Mifepristone.The preparation cell lysate, and with resisting-FAK (UBI ^TM, Lake Placid, N.Y.) monoclonal antibody immunity exhausts whole FAK albumen.Then, the FAK immunocomplex is carried out the SDS-polyacrylamide gel electrophoresis, and carry out immunoblotting with anti--FAK (A17) polyclonal antibody.Table 1 is presented at the photodensitometric quantitation that the radioautograph film is measured with " band light " unit at random, changes with the multiple of comparing the FAK protein level with irritation cell not.

Table 1: with the Mifepristone inductive FAK protein level of optical density(OD) flow measurement

Clone's numbering	????????FAKwt1		????????FAKwt3		??????????FAKwt4		?????????FAKwt5
									Mifepristone	??+	??-	??+	??-	??+	??-	??+	??-
Band Light unit	??2.00E+06	??1.00E+06	??8.00E+05	??5.00E+05	??2.00E+0 ??6	??5.00E+05	??3.00E+06	??5.00E+05
									Stimulate multiple	??2	??1	??2	??1	??4	??1	??6	??1

Observe FAK ^WTBut inducing cell is strong to the Mifepristone irritant reaction.

Above-mentioned condition can be interpreted as 96 hole ELISA forms at an easy rate.Table 2 has shown to be induced expresses FAK ^WTOr FAK ^Y397FProteic NIH3T3 clone.Catch FAK ^WTAnd FAK ^Y397FAlbumen is measured by whole FAK albumen of Mifepristone inductive or Tyrosine O-phosphate FAK ^Y397F

Table 2: measure the Mifepristone inductive FAK protein level that obtains with optical density(OD) 450

	??NIH3T3FAKwt		Letter (+)/(-) ratio of making an uproar	?????NIH3T3FAK ?????Y397F		Letter (+)/(-) ratio of making an uproar	The NIH3T3 carrier		Letter (+)/(-) ratio of making an uproar
										Mifepristone	??+	??-		??+	??-		??+	??-
Phosphoric acid FAKY397 catches	??1.69	??0.23	??7	??0.1	??0.12	??1	??0.08	??0.04	??2
										FAK albumen is caught	??1.94	??0.33	??6	??2.56	??0.46	??6	??0.21	??0.16	??1

Observe the NIH3T3 clone and produce intensive Tyrosine O-phosphate FAK ^Y397F, and FAK albumen signal to noise ratio be respectively～7 and～6.NIH3T3 tyrosine inefficacy clone and carrier NIH3T3 transfectant all prove the specificity of test to phosphoric acid FAKY397.

Table 3 is presented at the A431:FAK clone who analyzes under the optimal condition.

Table 3: measure the Mifepristone inductive FAK protein level that obtains with optical density(OD) 450

	??A431：FAKwt		Letter (+)/(-) ratio of making an uproar	????A31：FAK ????Y397F		Letter (+)/(-) ratio of making an uproar	A31: carrier		Letter (+)/(-) ratio of making an uproar
										Mifepristone	??+	??-		??+	??-		??+	??-
Phosphoric acid FAKY397 catches	??2.09	??0.13	??17	??0.13	??0.08	??1.4	??0.12	??0.11	??1.0
										FAK albumen is caught	??2.22	??0.21	??11	??2.31	??0.22	??11	??0.19	??0.17	??1.0

Under optimal condition, described A431:FAK clone produces intensive Tyrosine O-phosphate FAK ^Y397F, and FAK albumen signal to noise ratio be respectively～17 and～11.A431 tyrosine inefficacy clone and carrier A 431 transfectants all prove the specificity of test to phosphoric acid FAKY397.Proteic the inducing by changing test conditions of Tyrosine O-phosphate Y397 and FAK controlled, as the Mifepristone incubation time, and Mifepristone concentration, cell density and antibody concentration.

In A431 and other cell backgrounds, FAK activatory mechanism is studied.With A431:FAK ^WTThe not treated placement of cell or stimulate it with Mifepristone.At first, wash-out experiment (that is, will stimulate the cell of spend the night (16 hours) to cultivate for some time subsequently in the fresh growth medium of no Mifepristone with Mifepristone) shows that FAK albumen and Tyrosine O-phosphate Y397 level are time-dependent manner and reduce.Then, separate the A431:FAK that Mifepristone stimulates ^WTCell, and make it be suspended in the fresh growth medium 15 minutes, then, renewed vaccination in the culture dish of handling with tissue culture medium (TCM) (Petri dish) 4,24,48 and 72 hours.Table 4, experiment A shows the photodensitometric quantitation that " bandlight " the at random unit with the radioautograph film measures, and changes with the multiple that does not stimulate contrast to compare the FAK phosphorylation.

Table 4: with the FAK phosphorylation of photodensitometric quantitation mensuration

Experiment A

??A	???????????????????????????A431FAKwt
									4 hours	24 hours	48 hours	72 hours
Mifepristone	??-	??+	??+	??+	??+	??+
							Band Light unit	??5.7534	??57.832	??77.375	??71.033	??27.719	??92.834
The multiple of comparing with inducing cell not increases	??1	??10	??13	??12	??5	??16

Experiment B

??B	???????????????????A431FAKwt
									4 hours	24 hours	48 hours	72 hours
Mifepristone	?-	??+	??+	??+	??+	??+
							Band light unit	?20723	??44791	??89444	??65946	??11473	??82273
The multiple of comparing with inducing cell not increases	?1	??2	??4	??3	??1	??4

In experiment B, observe faint induce higher 4 times than untreated cell.But irritation cell being suspended after 15 minutes to adhere to plastics in fresh growth medium made phosphoric acid FAK in 4 hours ^Y397Reduce.In addition, allow the Mifepristone-inductive cell that suspends adhere to again 24 hours, can make Tyrosine O-phosphate FAK ^Y397Activation once more, this explanation FAK activate mechanism is complete.Consistent with the time-dependent manner reduction of FAK protein level, by 72 hours, Tyrosine O-phosphate FAK ^Y397Be reduced to without the stimulated control level.

Cell suspension causes FAK phosphorylation inactivation.Allow cell attachment that FAK is reactivated by the renewed vaccination suspension cell.Table 4 explanation is under the regulation system of above-mentioned discussion, and the FAK activate mechanism is complete.That is to say that the exogenous stimulation of Mifepristone causes the relevant FAK activation of machinery.

FAK inducibility of the present invention is successfully used to identify some FAK inhibitor based on the test of cell, comprises PP1 and Staurosporine.In addition, this test is used to measure the cytotoxicity of specific compound to the FAK express cell.In these experiments, make test compound and FAK inductive cell or not the inductive control cells contact.Table 5 shows when handling with the FAK inhibitor that increases concentration, the variation of the FAK phosphorylation under the OD450 unit.The multiple of FAK phosphorylation changed when table 6 showed with the photodensitometric quantitation of " band light " at random unit mensuration of radioautograph film and the increase of FAK inhibitor concentration.Therefore, test of the present invention can conveniently be used for the FAK drug development program.

As described in Figure 3, the FAK inducibility can be used for screening the FAK inhibitor based on the test of cell.10 μ m curves of FAK inhibitor carry out with 1/2 log10 dilution as shown in table 5.Measure and suppress per-cent, and calculate 50% (IC ₅₀) time inhibition concentration.In the T25 flask, inoculate A431:FAK ^WTCell to density is～80%, and not treated placement or stimulate with the 0.1nm Mifepristone is spent the night.Then, use the FAK inhibitor identical with table 5, with the 1/2 logarithm concentration identical with 10 μ m curves at 37 ℃, 5%CO ₂Processing was by stimulated cells 30 minutes.Prepare product of cell lysis then and measure total protein concentration by analysis of protein.Use Tyrosine O-phosphate FAK ^Y397Specific antibody, common Tyrosine O-phosphate pY20 antibody and anti-FAK (A17) antibody carry out the SDS-polyacrylamide gel electrophoresis and western analyzes to the adequate proteins of equivalent,

Table 5: measure the FAK phosphorylation that obtains with optical density(OD) 450

	Noise	FAK inhibitor+A431FAKwt
									??WT	??10μM	??3.3μM	??1.1μM	??0.37μM	??0.12μM	??0μM
Mifepristone	??-	??+	??+	??+	??+	??+	??+
								Phosphoric acid FAKY397	??0.28	??0.56	??0.98	??1.65	??2.21	??2.56	??2.77

??(OD450)
								Background subtraction	??0.000	??0.28	??0.70	??1.37	??1.93	??2.28	??2.49
The % contrast		??11	??28	??55	??78	??92	??100
								% suppresses		??89％	??72％	??45％	??22％	??8％	??0％

50% inhibition concentration of the FAK inhibitor that calculates is～0.93 μ M.

The multiple of FAK phosphorylation changed when table 6 showed with the photodensitometric quantitation of " band light " at random unit mensuration of radioautograph film and the increase of FAK inhibitor concentration.These experiments further illustrate the present invention's test in the calculated purposes of FAK drug discovery.

Table 6: with the FAK phosphorylation of photodensitometric quantitation mensuration

??A：	IB: anti-FAK (A17)
									Background	FAK inhibitor+A431FAKwt
	??WT	??10μM	??3.3μM	??1.1μM	??0.37μM	??0.12μM	??0μM
								Mifepristone	??-	??+	??+	??+	??+	??+	??+
Band Light unit	??30.501	??119.6	??100.2	??108.3	??128.1	??90.087	??82.4 ??14
								Background subtraction	??0	??89	??70	??78	??98	??60	??52
The % contrast		??172	??134	??150	??188	??115	??100
								% suppresses		??0％	??0％	??0％	??0％	??0％	??0％

??B：	IB: anti-FAKpY397
									Background	FAK inhibitor+A431FAKwt
	?WT	??10μM	??3.3μM	??1.1μM	??0.37μM	??0.12μM	??0μM
								Mifepristone	?-	??+	??+	??+	??+	??+	??+
Band Light unit	?0.34513	??3.9238	??10.44	??20.21	??45.27	??64.56	??68.022
								Background subtraction	?0	??4	??10	??20	??45	??64	??68
The % contrast		??5	??15	??29	??66	??95	??100
								% suppresses		??95％	??85％	??71％	??34％	??5％	??0％

??C：	IB: anti-pY20
									Background	FAK inhibitor+A431FAKwt
	??WT	??10μM	??3.3μM	??1.1μM	??0.37μM	??0.12μM	??0μ ??M
								Mifepristone	??-	??+	??+	??+	??+	??+	??+
Band light unit	??0.7577	??5.2206	??9.058	??21.51	??25.19	??26.323	??33.1 ??32
								Background subtraction	??0	??4	??8	??21	??24	??26	??32
The % contrast		??14	??26	??64	??75	??79	??100
								% suppresses		??86％	??74％	??36％	??25％	??21％	??0

As the contrast of albumen application of sample, the FAK protein content among the experiment A equates, and with FAK inhibitor incubation FAK is expressed without any influence.In experiment B, consistent with the IC50 that table 5 is reported, anti-FAK-pY397 trace shows the IC50 value of estimating in the 0.37-1.1 mu m range, and wherein 50% concentration is near 1.1 μ m.As test shown in the C, similar and consistent with the biology of FAK, estimated that with whole FAK phosphorylations that anti-pY20 immunoblotting is measured the inhibitor of IC50 scope at 1.1-3.3 μ m suppresses, wherein 50% inhibition concentration is near 3.3 μ m.

The external source regulation and control of FAK phosphorylation

By being purchased every hole inoculation 2.0 * 10 on fibronectin (FN) 96 orifice plates or homemade FN 96 orifice plates ⁵The ECM that cell obtains FAK stimulates.At 37 ℃, 5%CO ₂Allow cell attachment 15,30,60 or 90 minutes, thus induce integral protein to engage (engagement) and FAK phosphorylation subsequently.At RIPA lysis buffer (50mM Tris-HCl, pH7.4,1%NP-40,0.25% Sodium desoxycholate, 150mM NaCI, 1mM EDTA, 1mM Na ₃VO ₄, 1mM NaF and Complete of every 50ml solution ^TMNo EDTA proteinase inhibitor) preparation cell lysate in, and be transferred on 96 orifice plates of anti-FAK bag quilt to catch whole FAK albumen.Utilize common phosphotyrosine antibody Py54 to measure Tyrosine O-phosphate FAK albumen.Similarly, irritation cell on the flat board that is purchased matrigel bag quilt or on the homemade matrigel flat board.Analyze for western, allow cell on the flask that the flask or the self-control ECM of the ECM bag quilt that is purchased wrap quilt, adhere to the time as implied above.In the RIPA lysis buffer, prepare cell lysate, and utilize anti-FAK antibody or anti-phosphotyrosine antibody sedimentation (pull-down) or immunodetection all with Tyrosine O-phosphate FAK albumen.

On 96 orifice plates or T25/T75 flask, with cell (as A2058 metastatic human melanoma) be seeded to growth medium (DMEM 10%FBS, Pen/Strep/Glu) in, and make it, 5%CO at 37 ℃ ₂Adhered on the plastics that tissue culture medium (TCM) handles about 5 hours.Subsequently, at 37 ℃, 5%CO ₂Cell hunger in the hungry substratum of 0.1%FBS DMEM is spent the night, then, with the plain I of (maximum 100 μ M's) endotheliocyte, (maximum 100nM's) bombesin, or (maximum 800nM's) PMA handles irritation cell.At 37 ℃, 5%CO ₂, cellular exposure is not waited from 10 minutes to reaching 60 minutes in cytokine.Utilize anti-FAK specific antibody, catch or the whole FAK albumen of immunoprecipitation with the ELISA form.Then, detect Tyrosine O-phosphate FAK albumen with ELISA or Western form with common anti-FAK phosphotyrosine antibody.

Before cultivating hungry cell with the ﹠ beta 1 integrin antibody (4 ℃, 30 minutes) of different concns (1: 100 of 200 μ g/ml original liquid concentrations, 1: 330,1: 1000,1: 3,300 or 1: 10,000), with hungry cell (as A2058) precooling ensconce 4 ℃ 30 minutes.At 4 ℃, induced ﹠ beta 1 integrin bunch collection 30 minutes with 1: 500 dilution mountain sheep anti mouse two is anti-.Then, the full cell lysate of preparation in lysis buffer (10mM Tris-HCl, 5mMNaCl, 10mM EDTA, 2mM vanadic acid sodium, 1%NP40, proteinase inhibitor), and utilize anti-FAK antibody to make whole FAK protein immunization precipitations.Utilize anti-phosphotyrosine antibody Py54 to measure Tyrosine O-phosphate FAK albumen.

FAK ^WT With FAK mutant (not inducibility and Tet-On ^TM / Tet-Off ^TM System) clone

In RT-PCR, utilize primers F AK5 ' Bam:GGATCCATGGCAGCTGCTTACCTTGAC (SEQ ID NO:9) and FAK3 ' Bam:GGATCCTCACTCACTCAGTGTGGTCTCGTCTGCCCA (SEQ ID NO:10) from the T cell cdna library, to clone total length FAK ^WTCDNA, and determine sequence with accession number L13616 contrast.Utilize StratageneQuikChange ^TMThe site-directed mutagenesis test kit is to FAK ^WTTemplate is carried out site-directed mutagenesis.Be used for the plasmid that electroporation is gone into previously prepared HeLa of Clontech or HEK293tet-On/tet-Off clone in order to produce, with total length FAK ^WT, tyrosine inefficacy mutant (FAK ^Y397F) and kinases inefficacy mutant (FAK ^K454R) the cDNA subclone goes into the BamH1 site of pTRE:FLAG carrier.Prepare following configuration, and its electroporation is gone into HeLa Tet-Off, HeLa Tet-On, or 293Tet-On:pTRE:FLAG carrier, pTRE:FAK ^WTFLAG and pTRE:FAK ^Y397FFLAG.Select the G418 resistance clone, and in DMEM+100ug/ml G418 growth medium, breed.

FAK ^WT With FAK mutant (GeneSwitch ^TM System) clone

In RT-PCR, utilize above-mentioned primer from the T cell cdna library, to clone total length focal adhesion kinase (FAK ^WT) cDNA, and confirm sequence with accession number L13616 contrast.Utilize StratageneQuikChange ^TMThe site-directed mutagenesis test kit is to FAK ^WTTemplate is carried out site-directed mutagenesis.With total length FAK ^WT, tyrosine inefficacy mutant (FAK ^Y397F) and kinases inefficacy mutant (FAK ^K454R) the cDNA subclone goes into the BamHI-Apal site of pGeneV5/His-A plasmid.Dominant negative FAK dependency nonkinase (FRNK) cDNA is inserted the fragment subclone as Kpnl-Apal to go in the pGeneV5/His A-carrier box.Determine these plasmids by dna sequencing and restricted enzyme cutting analysis.Then, utilize the GeneJammer of Stratagene ^TMTransfection reagent is with these configurations and coding GeneSwitch ^TMProteic pSwitch configuration cotransfection is gone into NIH3T3, A431 or U87MG cell.Transfectant be mixed with 750 μ g/ml Zeocin and the antibiotic DMEM growth medium of 50 μ g/ml Totomycin (10%FBS, Pen/Strep/Glu) in the growth and be selected.Select Totomycin and Zeocin resistance clone, and propagation is screened in substratum being used at Western and RT-PCR.

A431FAK clone's screening

In the T25 flask, inoculation A431FAk ^WTAnd A431FAK ^Y397FBe cloned into degree of converging near 80%, unprocessed placement or handle spend the night (～16 hours) with 0.1nM Mifepristone part.Then, at RIPA lysis buffer (50mM Tris-HCl, pH7.4,1%NP-40,0.25% Sodium desoxycholate, 150mM NaCI, 1mM EDTA, 1mM Na ₃VO ₄, Complete in 1mM NaF and the every 50ml solution ^TMNo EDTA proteinase inhibitor) cracking A431 transfectant in, and analyze the total protein concentration of described cell lysate.Utilize standard western engram technology, with the total protein concentration of equivalent to FAK or FAK mutant, albumen and Tyrosine O-phosphate FAK ^Y397Carry out the western immunoblotting assay.The clone who selects test positive and show at least 3 times of induction ratio endogenous FAK level height comes optimization and develops the test of FAK based on cell.

Also the clone is screened and confirm by RT-PCR.Utilize sexamer at random, from A431FAK wild-type and mutant clon, separate and purifying kytoplasm mRNA transcript to be used for cDNA synthetic.Subsequently, utilize, carry out the polymerase chain reaction in cDNA library derived from the A431FAK transcript to N-terminal, inner FAK sequence and the special primer of FAK C-terminal.Also use these genes that increase, as the internal contrast of transfection and cDNA library quality to the special primer of microbiotic transcript Zeocin or Totomycin and GAPDH.

The FAK inducibility is based on the system optimization of cell

In the T25 flask, inoculate A431pGene ^Vector, A431FAK ^WT, A431FAK ^K454RAnd A431FAK ^Y397FBe cloned into degree of converging near 80%, not treated placement or with 0.1,10 or 100nM Mifepristone part handle spend the night (～16 hours).Then, cracking A431 transfectant in RIPA lysis buffer (as mentioned above), and analyze the total protein concentration of described cell lysate.Utilize the western engram technology of standard, carry out FAK or FAK mutant, albumen and Tyrosine O-phosphate FAK with the total protein concentration of equivalent ^Y397The western immunoblotting assay.In order to determine that optimized Mifepristone concentration is translated into the ELISA system from the western form, also optimization Mifepristone concentration and incubation time in the ELISA form.

In 96 hole U type base plates, with 1.2 * 10 ⁶The cell density inoculation A431pGene of cell/ml ^Vector, A431FAK ^WTAnd A431FAK ^Y397FThe clone.Before inducing, cell is placed 37 ℃, 5%CO with the Mifepristone part ₂6-8 hour.Then, the not treated placement of A431 clone or at 37 ℃, 5%CO ₂With 0.1,10,100 or the 1000nM Mifepristone handle～0.5,1.0,2.0,4.0 or 24.0 hours.Then, lysing cell in the RIPA lysis buffer, and 100 μ l cell lysates (45 μ g total protein) are transferred in 96 orifice plates.Using FAK specific antibody or Tyrosine O-phosphate FAK respectively ^Y397Catch FAK or FAK mutain and Tyrosine O-phosphate FAK on the mountain sheep anti mouse of antibody sandwich or the goat antirabbit flat board ^Y397In order to catch FAK or FAK mutain, for example, before hatching mountain sheep anti mouse flat board, that the mountain sheep anti mouse is dull and stereotyped with the anti-FAK of 0.5 μ g/ml (UBI) monoclonal antibody bag quilt with 100 μ l cell lysates (45 μ g total protein).Measuring the FAK or the FAK mutain of catching by anti-FAK (A17) polyclonal antibody detects.Similarly, by with the anti-FAKp[Y397 of 3.5 μ g/ml] the goat antirabbit flat board of polyclonal antibody bag quilt carries out Tyrosine O-phosphate FAK ^Y397Proteic seizure uses anti-FAK (UBI) monoclonal antibody to detect subsequently.

The parameter of the specific high throughput screening system of can optimization using, comprise estimate 96 orifice plates type (for example, anti-rabbit, albumin A or Protein G), capture antibodies concentration, detect antibody concentration, two anti-concentration of horseradish peroxidase (HRP) mark, cell density, the sealing damping fluid is (for example, SuperBlockBlocking TBS, the 3%BSA sealing) and the compound treatment time.

Utilizing the GSTFAK albumen in Y397 residue phosphorylation of purifying, at first the anti-FAKp[Y397 that increases with concentration] the anti-rabbit flat board in 96 holes of phosphoric acid specific antibody bag quilt determines optimized capture antibody concentration.Make the phosphoric acid specificity GSTFAK that catches ^Y397Antagonism FAKp[Y397] the antibody concentration chart to be to determine optimized capture antibodies concentration.Utilize this preliminary anti-FAKp[Y397] catch concentration, with other parameters (detecting and two anti-concentration cell density, sealing damping fluid) optimization, and format (formatted) is used for high flux screening (HTS).These parameters are most of optimised, or estimate simultaneously than the signal to noise ratio variation of relatively large conversion (permutation) by setting up 96 hole matrixs or intersecting.For example, with the anti-FAKp[Y397 of 3.5 μ g/ml] on the anti-rabbit flat board in 96 holes of capture antibodies bag quilt, in 96 pore matrix, set up detection that concentration increases and two anti- ^HRPTo detect the Tyrosine O-phosphate FAK that catches ^Y397Albumen, thereby the concentration of these antibody of optimization.Can on the albumin A flat board, repeat this experiment and/or intersect snr value than relatively large conversion, thereby make system optimization by changing sealing damping fluid or other parameters.

Confirm the test of FAK based on cell

In the T25 flask, inoculate A431FAK ^WTCell to degree of converging near 80%, not treated placement or handle spend the night (～16 hours) with 0.1nM Mifepristone part.At 37 ℃, 5%CO ₂, with 10ml PBS washing A431FAK ^WTNot inductive and inductive cell and be suspended in the 15ml growth medium (DMEM 10%FBS, Pen/Strep/Glu, 750 μ g/ml Zeocin, 50 μ g/ml Totomycin) 30,60,90 or 120 minutes.Then, lysing cell and analyze the total protein concentration of described cell lysate in RIPA lysis buffer (as mentioned above).Utilize standard western engram technology, carry out FAK or Tyrosine O-phosphate FAK with the total protein concentration of equivalent ^Y397The western immunoblotting assay.

In the T25 flask, inoculate A431FAK ^WTAnd A431FAK ^Y397FBe cloned into degree of converging near 80%, not treated placement or handle spend the night (～16 hours) with 0.1nM Mifepristone part.Then, the FAK inhibitor (half-logs of 10 μ M starting solns) that increases with concentration is handled A431FAK ^WTCell, described FAK inhibitor are to be that the test on basis is identified with FAK inducibility cell.Then, in the RIPA lysis buffer, prepare cell lysate, and the analyzing total protein concentration.Utilize standard western engram technology, carry out FAK, common Tyrosine O-phosphate FAK or Tyrosine O-phosphate FAK with the total protein concentration of equivalent ^Y397Proteic western immunoblotting assay.

Sequence table

＜110〉Pfizer Products Inc (Pfizer Products Inc.)

Roberts，Walter?Gregory

Ung，Ethan?James?Tekly

Whalen，Pamela?Mathews

＜120〉derivable focal adhesion kinase cell tests

<130>PC11699A

<160>10

<170>PatentIn?version?3.2

<210>1

<211>1052

<212>PRT

＜213〉people's wild-type FAK (not comprising V5 epi-position and His mark)

<400>1

Met?Ala?Ala?Ala?Tyr?Leu?Asp?Pro?Asn?Leu?Asn?His?Thr?Pro?Asn?Ser

1???????????????5???????????????????10??????????????????15

Ser?Thr?Lys?Thr?His?Leu?Gly?Thr?Gly?Met?Glu?Arg?Ser?Pro?Gly?Ala

20??????????????????25??????????????????30

Met?Glu?Arg?Val?Leu?Lys?Val?Phe?His?Tyr?Phe?Glu?Ser?Asn?Ser?Glu

35??????????????????40??????????????????45

Pro?Thr?Thr?Trp?Ala?Ser?Ile?Ile?Arg?His?Gly?Asp?Ala?Thr?Asp?Val

50??????????????????55??????????????????60

Arg?Gly?Ile?Ile?Gln?Lys?Ile?Val?Asp?Ser?His?Lys?Val?Lys?His?Val

65??????????????????70??????????????????75??????????????????80

Ala?Cys?Tyr?Gly?Phe?Arg?Leu?Ser?His?Leu?Arg?Ser?Glu?Glu?Val?His

85??????????????????90??????????????????95

Trp?Leu?His?Val?Asp?Met?Gly?Val?Ser?Ser?Val?Arg?Glu?Lys?Tyr?Glu

100?????????????????105?????????????????110

Leu?Ala?His?Pro?Pro?Glu?Glu?Trp?Lys?Tyr?Glu?Leu?Arg?Ile?Arg?Tyr

115?????????????????120?????????????????125

Leu?Pro?Lys?Gly?Phe?Leu?Asn?Gln?Phe?Thr?Glu?Asp?Lys?Pro?Thr?Leu

130?????????????????135?????????????????140

Asn?Phe?Phe?Tyr?Gln?Gln?Val?Lys?Ser?Asp?Tyr?Met?Leu?Glu?Ile?Ala

145?????????????????150?????????????????155?????????????????160

Asp?Gln?Val?Asp?Gln?Glu?Ile?Ala?Leu?Lys?Leu?Gly?Cys?Leu?Glu?Ile

165?????????????????170?????????????????l75

Arg?Arg?Ser?Tyr?Trp?Glu?Met?Arg?Gly?Asn?Ala?Leu?Glu?Lys?Lys?Ser

180?????????????????185?????????????????190

Asn?Tyr?Glu?Val?Leu?Glu?Lys?Asp?Val?Gly?Leu?Lys?Arg?Phe?Phe?Pro

195?????????????????200?????????????????205

Lys?Ser?Leu?Leu?Asp?Ser?Val?Lys?Ala?Lys?Thr?Leu?Arg?Lys?Leu?Ile

210?????????????????215?????????????????220

Gln?Gln?Thr?Phe?Arg?Gln?Phe?Ala?Asn?Leu?Asn?Arg?Glu?Glu?Ser?Ile

225?????????????????230?????????????????235?????????????????240

Leu?Lys?Phe?Phe?Glu?Ile?Leu?Ser?Pro?Val?Tyr?Arg?Phe?Asp?Lys?Glu

245?????????????????250?????????????????255

Cys?Phe?Lys?Cys?Ala?Leu?Gly?Ser?Ser?Trp?Ile?Ile?Ser?Val?Glu?Leu

260?????????????????265?????????????????270

Ala?Ile?Gly?Pro?Glu?Glu?Gly?Ile?Ser?Tyr?Leu?Thr?Asp?Lys?Gly?Cys

275?????????????????280?????????????????285

Asn?Pro?Thr?His?Leu?Ala?Asp?Phe?Thr?Gln?Val?Gln?Thr?Ile?Gln?Tyr

290?????????????????295?????????????????300

Ser?Asn?Ser?Glu?Asp?Lys?Asp?Arg?Lys?Gly?Met?Leu?Gln?Leu?Lys?Ile

305?????????????????310?????????????????315?????????????????320

Ala?Gly?Ala?Pro?Glu?Pro?Leu?Thr?Val?Thr?Ala?Pro?Ser?Leu?Thr?Ile

325?????????????????330?????????????????335

Ala?Glu?Asn?Met?Ala?Asp?Leu?Ile?Asp?Gly?Tyr?Cys?Arg?Leu?Val?Asn

340?????????????????345?????????????????350

Gly?Thr?Ser?Gln?Ser?Phe?Ile?Ile?Arg?Pro?Gln?Lys?Glu?Gly?Glu?Arg

355?????????????????360?????????????????365

Ala?Leu?Pro?Ser?Ile?Pro?Lys?Leu?Ala?Asn?Ser?Glu?Lys?Gln?Gly?Met

370?????????????????375?????????????????380

Arg?Thr?His?Ala?Val?Ser?Val?Ser?Glu?Thr?Asp?Asp?Tyr?Ala?Glu?Ile

385?????????????????390?????????????????395?????????????????400

Ile?Asp?Glu?Glu?Asp?Thr?Tyr?Thr?Met?Pro?Ser?Thr?Arg?Asp?Tyr?Glu

405?????????????????410?????????????????415

Ile?Gln?Arg?Glu?Arg?Ile?Glu?Leu?Gly?Arg?Cys?Ile?Gly?Glu?Gly?Gln

420?????????????????425?????????????????430

Phe?Gly?Asp?Val?His?Gln?Gly?Ile?Tyr?Met?Ser?Pro?Glu?Asn?Pro?Ala

435?????????????????440?????????????????445

Leu?Ala?Val?Ala?Ile?Lys?Thr?Cys?Lys?Asn?Cys?Thr?Ser?Asp?Ser?Val

450?????????????????455?????????????????460

Arg?Glu?Lys?Phe?Leu?Gln?Glu?Ala?Leu?Thr?Met?Arg?Gln?Phe?Asp?His

465?????????????????470?????????????????475?????????????????480

Pro?His?Ile?Val?Lys?Leu?Ile?Gly?Val?Ile?Thr?Glu?Asn?Pro?Val?Trp

485?????????????????490?????????????????495

Ile?Ile?Met?Glu?Leu?Cys?Thr?Leu?Gly?Glu?Leu?Arg?Ser?Phe?Leu?Gln

500?????????????????505?????????????????510

Val?Arg?Lys?Tyr?Ser?Leu?Asp?Leu?Ala?Ser?Leu?Ile?Leu?Tyr?Ala?Tyr

515?????????????????520?????????????????525

Gln?Leu?Ser?Thr?Ala?Leu?Ala?Tyr?Leu?Glu?Ser?Lys?Arg?Phe?Val?His

530?????????????????535?????????????????540

Arg?Asp?Ile?Ala?Ala?Arg?Asn?Val?Leu?Val?Ser?Ser?Asn?Asp?Cys?Val

545?????????????????550?????????????????555?????????????????560

Lys?Leu?Gly?Asp?Phe?Gly?Leu?Ser?Arg?Tyr?Met?Glu?Asp?Ser?Thr?Tyr

565?????????????????570?????????????????575

Tyr?Lys?Ala?Ser?Lys?Gly?Lys?Leu?Pro?Ile?Lys?Trp?Met?Ala?Pro?Glu

580?????????????????585?????????????????590

Ser?Ile?Asn?Phe?Arg?Arg?Phe?Thr?Ser?Ala?Ser?Asp?Val?Trp?Met?Phe

595?????????????????600?????????????????605

Gly?Val?Cys?Met?Trp?Glu?Ile?Leu?Met?His?Gly?Val?Lys?Pro?Phe?Gln

610?????????????????615?????????????????620

Gly?Val?Lys?Asn?Asn?Asp?Val?Ile?Gly?Arg?Ile?Glu?Asn?Gly?Glu?Arg

625?????????????????630?????????????????635?????????????????640

Leu?Pro?Met?Pro?Pro?Asn?Cys?Pro?Pro?Thr?Leu?Tyr?Ser?Leu?Met?Thr

645?????????????????650?????????????????655

Lys?Cys?Trp?Ala?Tyr?Asp?Pro?Ser?Arg?Arg?Pro?Arg?Phe?Thr?Glu?Leu

660?????????????????665?????????????????670

Lys?Ala?Gln?Leu?Ser?Thr?Ile?Leu?Glu?Glu?Glu?Lys?Ala?Gln?Gln?Glu

675?????????????????680?????????????????685

Glu?Arg?Met?Arg?Met?Glu?Ser?Arg?Arg?Gln?Ala?Thr?Val?Ser?Trp?Asp

690?????????????????695?????????????????700

Ser?Gly?Gly?Ser?Asp?Glu?Ala?Pro?Pro?Lys?Pro?Ser?Arg?Pro?Gly?Tyr

705?????????????????710?????????????????715?????????????????720

Pro?Ser?Pro?Arg?Ser?Ser?Glu?Gly?Phe?Tyr?Pro?Ser?Pro?Gln?His?Met

725?????????????????730?????????????????735

Val?Gln?Thr?Asn?His?Tyr?Gln?Val?Ser?Gly?Tyr?Pro?Gly?Ser?His?Gly

740?????????????????745?????????????????750

Ile?Thr?Ala?Met?Ala?Gly?Ser?Ile?Tyr?Pro?Gly?Gln?Ala?Ser?Leu?Leu

755?????????????????760?????????????????765

Asp?Gln?Thr?Asp?Ser?Trp?Asn?His?Arg?Pro?Gln?Glu?Ile?Ala?Met?Trp

770?????????????????775?????????????????780

Gln?Pro?Asn?Val?Glu?Asp?Ser?Thr?Val?Leu?Asp?Leu?Arg?Gly?Ile?Gly

785?????????????????790?????????????????795?????????????????800

Gln?Val?Leu?Pro?Thr?His?Leu?Met?Glu?Glu?Arg?Leu?Ile?Arg?Gln?Gln

805?????????????????810?????????????????815

Gln?Glu?Met?Glu?Glu?Asp?Gln?Arg?Trp?Leu?Glu?Lys?Glu?Glu?Arg?Phe

820?????????????????825?????????????????830

Leu?Lys?Pro?Asp?Val?Arg?Leu?Ser?Arg?Gly?Ser?Ile?Asp?Arg?Glu?Asp

835?????????????????840?????????????????845

Gly?Ser?Leu?Gln?Gly?Pro?Ile?Gly?Asn?Gln?His?Ile?Tyr?Gln?Pro?Val

850?????????????????855?????????????????860

Gly?Lys?Pro?Asp?Pro?Ala?Ala?Pro?Pro?Lys?Lys?Pro?Pro?Arg?Pro?Gly

865?????????????????870?????????????????875?????????????????880

Ala?Pro?Gly?His?Leu?Gly?Ser?Leu?Ala?Ser?Leu?Ser?Ser?Pro?Ala?Asp

885?????????????????890?????????????????895

Ser?Tyr?Asn?Glu?Gly?Val?Lys?Leu?Gln?Pro?Gln?Glu?Ile?Ser?Pro?Pro

900?????????????????905?????????????????910

Pro?Thr?Ala?Asn?Leu?Asp?Arg?Ser?Asn?Asp?Lys?Val?Tyr?Glu?Asn?Val

915?????????????????920?????????????????925

Thr?Gly?Leu?Val?Lys?Ala?Val?Ile?Glu?Met?Ser?Ser?Lys?Ile?Gln?Pro

930?????????????????935?????????????????940

Ala?Pro?Pro?Glu?Glu?Tyr?Val?Pro?Met?Val?Lys?Glu?Val?Gly?Leu?Ala

945?????????????????950?????????????????955?????????????????960

Leu?Arg?Thr?Leu?Leu?Ala?Thr?Val?Asp?Glu?Thr?Ile?Pro?Leu?Leu?Pro

965?????????????????970?????????????????975

Ala?Ser?Thr?His?Arg?Glu?Ile?Glu?Met?Ala?Gln?Lys?Leu?Leu?Asn?Ser

980?????????????????985?????????????????990

Asp?Leu?Gly?Glu?Leu?Ile?Asn?Lys?Met?Lys?Leu?Ala?Gln?Gln?Tyr?Val

995?????????????????1000?????????????????1005

Met?Thr?Ser?Leu?Gln?Gln?Glu?Tyr?Lys?Lys?Gln?Met?Leu?Thr?Ala

1010????????????????1015????????????????1020

Ala?His?Ala?Leu?Ala?Val?Asp?Ala?Lys?Asn?Leu?Leu?Asp?Val?Ile

1025????????????????1030????????????????1035

Asp?Gln?Ala?Arg?Leu?Lys?Met?Leu?Gly?Gln?Thr?Arg?Pro?His

1040????????????????1045????????????????1050

<210>2

<211>1052

<212>PRT

＜213〉human FAK Y397F mutant (not comprising V5 epi-position and His mark)

<400>2

Met?Ala?Ala?Ala?Tyr?Leu?Asp?Pro?Asn?Leu?Asn?His?Thr?Pro?Asn?Ser

1???????????????5???????????????????10??????????????????15

Ser?Thr?Lys?Thr?His?Leu?Gly?Thr?Gly?Met?Glu?Arg?Ser?Pro?Gly?Ala

20??????????????????25??????????????????30

Met?Glu?Arg?Val?Leu?Lys?Val?Phe?His?Tyr?Phe?Glu?Ser?Asn?Ser?Glu

35??????????????????40??????????????????45

Pro?Thr?Thr?Trp?Ala?Ser?Ile?Ile?Arg?His?Gly?Asp?Ala?Thr?Asp?Val

50??????????????????55??????????????????60

Arg?Gly?Ile?Ile?Gln?Lys?Ile?Val?Asp?Ser?His?Lys?Val?Lys?His?Val

65??????????????????70??????????????????75??????????????????80

Ala?Cys?Tyr?Gly?Phe?Arg?Leu?Ser?His?Leu?Arg?Ser?Glu?Glu?Val?His

85??????????????????90??????????????????95

Trp?Leu?His?Val?Asp?Met?Gly?Val?Ser?Ser?Val?Arg?Glu?Lys?Tyr?Clu

100?????????????????105?????????????????110

Leu?Ala?His?Pro?Pro?Glu?Glu?Trp?Lys?Tyr?Glu?Leu?Arg?Ile?Arg?Tyr

115?????????????????120?????????????????125

Leu?Pro?Lys?Gly?Phe?Leu?Asn?Gln?Phe?Thr?Glu?Asp?Lys?Pro?Thr?Leu

130?????????????????135?????????????????140

Asn?Phe?Phe?Tyr?Gln?Gln?Val?Lys?Ser?Asp?Tyr?Met?Leu?Glu?Ile?Ala

145?????????????????150?????????????????155?????????????????160

Asp?Gln?Val?Asp?Gln?Glu?Ile?Ala?Leu?Lys?Leu?Gly?Cys?Leu?Glu?Ile

165?????????????????170?????????????????175

Arg?Arg?Ser?Tyr?Trp?Glu?Met?Arg?Gly?Asn?Ala?Leu?Glu?Lys?Lys?Ser

180?????????????????185?????????????????190

Asn?Tyr?Glu?Val?Leu?Glu?Lys?Asp?Val?Gly?Leu?Lys?Arg?Phe?Phe?Pro

195?????????????????200?????????????????205

Lys?Ser?Leu?Leu?Asp?Ser?Val?Lys?Ala?Lys?Thr?Leu?Arg?Lys?Leu?Ile

210?????????????????215?????????????????220

Gln?Gln?Thr?Phe?Arg?Gln?Phe?Ala?Asn?Leu?Asn?Arg?Glu?Glu?Ser?Ile

225?????????????????230?????????????????235?????????????????240

Leu?Lys?Phe?Phe?Glu?Ile?Leu?Ser?Pro?Val?Tyr?Arg?Phe?Asp?Lys?Glu

245?????????????????250?????????????????255

Cys?Phe?Lys?Cys?Ala?Leu?Gly?Ser?Ser?Trp?Ile?Ile?Ser?Val?Glu?Leu

260?????????????????265?????????????????270

Ala?Ile?Gly?Pro?Glu?Glu?Gly?Ile?Ser?Tyr?Leu?Thr?Asp?Lys?Gly?Cys

275?????????????????280?????????????????285

Asn?Pro?Thr?His?Leu?Ala?Asp?Phe?Thr?Gln?Val?Gln?Thr?Ile?Gln?Tyr

290?????????????????295?????????????????300

Ser?Asn?Ser?Glu?Asp?Lys?Asp?Arg?Lys?Gly?Met?Leu?Gln?Leu?Lys?Ile

305?????????????????310?????????????????315?????????????????320

Ala?Gly?Ala?Pro?Glu?Pro?Leu?Thr?Val?Thr?Ala?Pro?Ser?Leu?Thr?Ile

325?????????????????330?????????????????335

Ala?Glu?Asn?Met?Ala?Asp?Leu?Ile?Asp?Gly?Tyr?Cys?Arg?Leu?Val?Asn

340?????????????????345?????????????????350

Gly?Thr?Ser?Gln?Ser?Phe?Ile?Ile?Arg?Pro?Gln?Lys?Glu?Gly?Glu?Arg

355?????????????????360?????????????????365

Ala?Leu?Pro?Ser?Ile?Pro?Lys?Leu?Ala?Asn?Ser?Glu?Lys?Gln?Gly?Met

370?????????????????375?????????????????380

Arg?Thr?His?Ala?Val?Ser?Val?Ser?Glu?Thr?Asp?Asp?Phe?Ala?Glu?Ile

385?????????????????390?????????????????395?????????????????400

Ile?Asp?Glu?Glu?Asp?Thr?Tyr?Thr?Met?Pro?Ser?Thr?Arg?Asp?Tyr?Glu

405?????????????????410?????????????????415

Ile?Gln?Arg?Glu?Arg?Ile?Glu?Leu?Gly?Arg?Cys?Ile?Gly?Glu?Gly?Gln

420?????????????????425?????????????????430

Phe?Gly?Asp?Val?His?Gln?Gly?Ile?Tyr?Met?Ser?Pro?Glu?Asn?Pro?Ala

435?????????????????440?????????????????445

Leu?Ala?Val?Ala?Ile?Lys?Thr?Cys?Lys?Asn?Cys?Thr?Ser?Asp?Ser?Val

450?????????????????455?????????????????460

Arg?Glu?Lys?Phe?Leu?Gln?Glu?Ala?Leu?Thr?Met?Arg?Gln?Phe?Asp?His

465?????????????????470?????????????????475?????????????????480

Pro?His?Ile?Val?Lys?Leu?Ile?Gly?Val?Ile?Thr?Glu?Asn?Pro?Val?Trp

485?????????????????490?????????????????495

Ile?Ile?Met?Glu?Leu?Cys?Thr?Leu?Gly?Glu?Leu?Arg?Ser?Phe?Leu?Gln

500?????????????????505?????????????????510

Val?Arg?Lys?Tyr?Ser?Leu?Asp?Leu?Ala?Ser?Leu?Ile?Leu?Tyr?Ala?Tyr

515?????????????????520?????????????????525

Gln?Leu?Ser?Thr?Ala?Leu?Ala?Tyr?Leu?Glu?Ser?Lys?Arg?Phe?Val?His

530?????????????????535?????????????????540

Arg?Asp?Ile?Ala?Ala?Arg?Asn?Val?Leu?Val?Ser?Ser?Asn?Asp?Cys?Val

545?????????????????550?????????????????555?????????????????560

Lys?Leu?Gly?Asp?Phe?Gly?Leu?Ser?Arg?Tyr?Met?Glu?Asp?Ser?Thr?Tyr

565?????????????????570?????????????????575

Tyr?Lys?Ala?Ser?Lys?Gly?Lys?Leu?Pro?Ile?Lys?Trp?Met?Ala?Pro?Glu

580?????????????????585?????????????????590

Ser?Ile?Asn?Phe?Arg?Arg?Phe?Thr?Ser?Ala?Ser?Asp?Val?Trp?Met?Phe

595?????????????????600?????????????????605

Gly?Val?Cys?Met?Trp?Glu?Ile?Leu?Met?His?Gly?Val?Lys?Pro?Phe?Gln

610?????????????????615?????????????????620

Gly?Val?Lys?Asn?Asn?Asp?Val?Ile?Gly?Arg?Ile?Glu?Asn?Gly?Glu?Arg

625?????????????????630?????????????????635?????????????????640

Leu?Pro?Met?Pro?Pro?Asn?Cys?Pro?Pro?Thr?Leu?Tyr?Ser?Leu?Met?Thr

645?????????????????650?????????????????655

Lys?Cys?Trp?Ala?Tyr?Asp?Pro?Ser?Arg?Arg?Pro?Arg?Phe?Thr?Glu?Leu

660?????????????????665?????????????????670

Lys?Ala?Gln?Leu?Ser?Thr?Ile?Leu?Glu?Glu?Glu?Lys?Ala?Gln?Gln?Glu

675?????????????????680?????????????????685

Glu?Arg?Met?Arg?Met?Glu?Ser?Arg?Arg?Gln?Ala?Thr?Val?Ser?Trp?Asp

690?????????????????695?????????????????700

Ser?Gly?Gly?Ser?Asp?Glu?Ala?Pro?Pro?Lys?Pro?Ser?Arg?Pro?Gly?Tyr

705?????????????????710?????????????????715?????????????????720

Pro?Ser?Pro?Arg?Ser?Ser?Glu?Gly?Phe?Tyr?Pro?Ser?Pro?Gln?His?Met

725?????????????????730?????????????????735

Val?Gln?Thr?Asn?His?Tyr?Gln?Val?Ser?Gly?Tyr?Pro?Gly?Ser?His?Gly

740?????????????????745?????????????????750

Ile?Thr?Ala?Met?Ala?Gly?Ser?Ile?Tyr?Pro?Gly?Gln?Ala?Ser?Leu?Leu

755?????????????????760?????????????????765

Asp?Gln?Thr?Asp?Ser?Trp?Asn?His?Arg?Pro?Gln?Glu?Ile?Ala?Met?Trp

770?????????????????775?????????????????780

Gln?Pro?Asn?Val?Glu?Asp?Ser?Thr?Val?Leu?Asp?Leu?Arg?Gly?Ile?Gly

785?????????????????790?????????????????795?????????????????800

Gln?Val?Leu?Pro?Thr?His?Leu?Met?Glu?Glu?Arg?Leu?Ile?Arg?Gln?Gln

805?????????????????810?????????????????815

Gln?Glu?Met?Glu?Glu?Asp?Gln?Arg?Trp?Leu?Glu?Lys?Glu?Glu?Arg?Phe

820?????????????????825?????????????????830

Leu?Lys?Pro?Asp?Val?Arg?Leu?Ser?Arg?Gly?Ser?Ile?Asp?Arg?Glu?Asp

835?????????????????840?????????????????845

Gly?Ser?Leu?Gln?Gly?Pro?Ile?Gly?Asn?Gln?His?Ile?Tyr?Gln?Pro?Val

850?????????????????855?????????????????860

Gly?Lys?Pro?Asp?Pro?Ala?Ala?Pro?Pro?Lys?Lys?Pro?Pro?Arg?Pro?Gly

865?????????????????870?????????????????875?????????????????880

Ala?Pro?Gly?His?Leu?Gly?Ser?Leu?Ala?Ser?Leu?Ser?Ser?Pro?Ala?Asp

885?????????????????890?????????????????895

Ser?Tyr?Asn?Glu?Gly?Val?Lys?Leu?Gln?Pro?Gln?Glu?Ile?Ser?Pro?Pro

900?????????????????905?????????????????910

Pro?Thr?Ala?Asn?Leu?Asp?Arg?Ser?Asn?Asp?Lys?Val?Tyr?Glu?Asn?Val

915?????????????????920?????????????????925

Thr?Gly?Leu?Val?Lys?Ala?Val?Ile?Glu?Met?Ser?Ser?Lys?Ile?Gln?Pro

930?????????????????935?????????????????940

Ala?Pro?Pro?Glu?Glu?Tyr?Val?Pro?Met?Val?Lys?Glu?Val?Gly?Leu?Ala

945?????????????????950?????????????????955?????????????????960

Leu?Arg?Thr?Leu?Leu?Ala?Thr?Val?Asp?Glu?Thr?Ile?Pro?Leu?Leu?Pro

965?????????????????970?????????????????975

Ala?Ser?Thr?His?Arg?Glu?Ile?Glu?Met?Ala?Gln?Lys?Leu?Leu?Asn?Ser

980?????????????????985?????????????????990

Asp?Leu?Gly?Glu?Leu?Ile?Asn?Lys?Met?Lys?Leu?Ala?Gln?Gln?Tyr?Val

995?????????????????1000????????????????1005

Met?Thr?Ser?Leu?Gln?Gln?Glu?Tyr?Lys?Lys?Gln?Met?Leu?Thr?Ala

1010????????????????1015????????????????1020

Ala?His?Ala?Leu?Ala?Val?Asp?Ala?Lys?Asn?Leu?Leu?Asp?Val?Ile

1025????????????????1030????????????????1035

Asp?Gln?Ala?Arg?Leu?Lys?Met?Leu?Gly?Gln?Thr?Arg?Pro?His

1040????????????????1045????????????????1050

<210>3

<211>1052

<212>PRT

＜213〉human FAK K454R mutant (not comprising V5 epi-position and His mark)

<400>3

Met?Ala?Ala?Ala?Tyr?Leu?Asp?Pro?Asn?Leu?Asn?His?Thr?Pro?Asn?Ser

1???????????????5???????????????????10??????????????????15

Ser?Thr?Lys?Thr?His?Leu?Gly?Thr?Gly?Met?Glu?Arg?Ser?Pro?Gly?Ala

20??????????????????25??????????????????30

Met?Glu?Arg?Val?Leu?Lys?Val?Phe?His?Tyr?Phe?Glu?Ser?Asn?Ser?Glu

35??????????????????40??????????????????45

Pro?Thr?Thr?Trp?Ala?Ser?Ile?Ile?Arg?His?Gly?Asp?Ala?Thr?Asp?Val

50??????????????????55??????????????????60

Arg?Gly?Ile?Ile?Gln?Lys?Ile?Val?Asp?Ser?His?Lys?Val?Lys?His?Val

65??????????????????70??????????????????75??????????????????80

Ala?Cys?Tyr?Gly?Phe?Arg?Leu?Ser?His?Leu?Arg?Ser?Glu?Glu?Val?His

85??????????????????90??????????????????95

Trp?Leu?His?Val?Asp?Met?Gly?Val?Ser?Ser?Val?Arg?Glu?Lys?Tyr?Glu

100?????????????????105?????????????????110

Leu?Ala?His?Pro?Pro?Glu?Glu?Trp?Lys?Tyr?Glu?Leu?Arg?Ile?Arg?Tyr

115?????????????????120?????????????????125

Leu?Pro?Lys?Gly?Phe?Leu?Asn?Gln?Phe?Thr?Glu?Asp?Lys?Pro?Thr?Leu

130?????????????????135?????????????????140

Asn?Phe?Phe?Tyr?Gln?Gln?Val?Lys?Ser?Asp?Tyr?Met?Leu?Glu?Ile?Ala

145?????????????????150?????????????????155?????????????????160

Asp?Gln?Val?Asp?Gln?Glu?Ile?Ala?Leu?Lys?Leu?Gly?Cys?Leu?Glu?Ile

165?????????????????170?????????????????175

Arg?Arg?Ser?Tyr?Trp?Glu?Met?Arg?Gly?Asn?Ala?Leu?Glu?Lys?Lys?Ser

180?????????????????185?????????????????190

Asn?Tyr?Glu?Val?Leu?Glu?Lys?Asp?Val?Gly?Leu?Lys?Arg?Phe?Phe?Pro

195?????????????????200?????????????????205

Lys?Ser?Leu?Leu?Asp?Ser?Val?Lys?Ala?Lys?Thr?Leu?Arg?Lys?Leu?Ile

210?????????????????215?????????????????220

Gln?Gln?Thr?Phe?Arg?Gln?Phe?Ala?Asn?Leu?Asn?Arg?Glu?Glu?Ser?Ile

225?????????????????230?????????????????235?????????????????240

Leu?Lys?Phe?Phe?Glu?Ile?Leu?Ser?Pro?Val?Tyr?Arg?Phe?Asp?Lys?Glu

245?????????????????250?????????????????255

Cys?Phe?Lys?Cys?Ala?Leu?Gly?Ser?Ser?Trp?Ile?Ile?Ser?Val?Glu?Leu

260?????????????????265?????????????????270

Ala?Ile?Gly?Pro?Glu?Glu?Gly?Ile?Ser?Tyr?Leu?Thr?Asp?Lys?Gly?Cys

275?????????????????280?????????????????285

Asn?Pro?Thr?His?Leu?Ala?Asp?Phe?Thr?Gln?Val?Gln?Thr?Ile?Gln?Tyr

290?????????????????295?????????????????300

Ser?Asn?Ser?Glu?Asp?Lys?Asp?Arg?Lys?Gly?Met?Leu?Gln?Leu?Lys?Ile

305?????????????????310?????????????????315?????????????????320

Ala?Gly?Ala?Pro?Glu?Pro?Leu?Thr?Val?Thr?Ala?Pro?Ser?Leu?Thr?Ile

325?????????????????330?????????????????335

Ala?Glu?Asn?Met?Ala?Asp?Leu?Ile?Asp?Gly?Tyr?Cys?Arg?Leu?Val?Asn

340?????????????????345?????????????????350

Gly?Thr?Ser?Gln?Ser?Phe?Ile?Ile?Arg?Pro?Gln?Lys?Glu?Gly?Glu?Arg

355?????????????????360?????????????????365

Ala?Leu?Pro?Ser?Ile?Pro?Lys?Leu?Ala?Asn?Ser?Glu?Lys?Gln?Gly?Met

370?????????????????375?????????????????380

Arg?Thr?His?Ala?Val?Ser?Val?Ser?Glu?Thr?Asp?Asp?Tyr?Ala?Glu?Ile

385?????????????????390?????????????????395?????????????????400

Ile?Asp?Glu?Glu?Asp?Thr?Tyr?Thr?Met?Pro?Ser?Thr?Arg?Asp?Tyr?Glu

405?????????????????410?????????????????415

Ile?Gln?Arg?Glu?Arg?Ile?Glu?Leu?Gly?Arg?Cys?Ile?Gly?Glu?Gly?Gln

420?????????????????425?????????????????430

Phe?Gly?Asp?Val?His?Gln?Gly?Ile?Tyr?Met?Ser?Pro?Glu?Asn?Pro?Ala

435?????????????????440?????????????????445

Leu?Ala?Val?Ala?Ile?Arg?Thr?Cys?Lys?Asn?Cys?Thr?Ser?Asp?Ser?Val

450?????????????????455?????????????????460

Arg?Glu?Lys?Phe?Leu?Gln?Glu?Ala?Leu?Thr?Met?Arg?Gln?Phe?Asp?His

465?????????????????470?????????????????475?????????????????480

Pro?His?Ile?Val?Lys?Leu?Ile?Gly?Val?Ile?Thr?Glu?Asn?Pro?Val?Trp

485?????????????????490?????????????????495

Ile?Ile?Met?Glu?Leu?Cys?Thr?Leu?Gly?Glu?Leu?Arg?Ser?Phe?Leu?Gln

500?????????????????505?????????????????510

Val?Arg?Lys?Tyr?Ser?Leu?Asp?Leu?Ala?Ser?Leu?Ile?Leu?Tyr?Ala?Tyr

515?????????????????520?????????????????525

Gln?Leu?Ser?Thr?Ala?Leu?Ala?Tyr?Leu?Glu?Ser?Lys?Arg?Phe?Val?His

530?????????????????535?????????????????540

Arg?Asp?Ile?Ala?Ala?Arg?Asn?Val?Leu?Val?Ser?Ser?Asn?Asp?Cys?Val

545?????????????????550?????????????????555?????????????????560

Lys?Leu?Gly?Asp?Phe?Gly?Leu?Ser?Arg?Tyr?Met?Glu?Asp?Ser?Thr?Tyr

565?????????????????570?????????????????575

Tyr?Lys?Ala?Ser?Lys?Gly?Lys?Leu?Pro?Ile?Lys?Trp?Met?Ala?Pro?Glu

580?????????????????585?????????????????590

Ser?Ile?Asn?Phe?Arg?Arg?Phe?Thr?Ser?Ala?Ser?Asp?Val?Trp?Met?Phe

595?????????????????600?????????????????605

Gly?Val?Cys?Met?Trp?Glu?Ile?Leu?Met?His?Gly?Val?Lys?Pro?Phe?Gln

610?????????????????615?????????????????620

Gly?Val?Lys?Asn?Asn?Asp?Val?Ile?Gly?Arg?Ile?Glu?Asn?Gly?Glu?Arg

625?????????????????630?????????????????635?????????????????640

Leu?Pro?Met?Pro?Pro?Asn?Cys?Pro?Pro?Thr?Leu?Tyr?Ser?Leu?Met?Thr

645?????????????????650?????????????????655

Lys?Cys?Trp?Ala?Tyr?Asp?Pro?Ser?Arg?Arg?Pro?Arg?Phe?Thr?Glu?Leu

660?????????????????665?????????????????670

Lys?Ala?Gln?Leu?Ser?Thr?Ile?Leu?Glu?Glu?Glu?Lys?Ala?Gln?Gln?Glu

675?????????????????680?????????????????685

Glu?Arg?Met?Arg?Met?Glu?Ser?Arg?Arg?Gln?Ala?Thr?Val?Ser?Trp?Asp

690?????????????????695?????????????????700

Ser?Gly?Gly?Ser?Asp?Glu?Ala?Pro?Pro?Lys?Pro?Ser?Arg?Pro?Gly?Tyr

705?????????????????710?????????????????715?????????????????720

Pro?Ser?Pro?Arg?Ser?Ser?Glu?Gly?Phe?Tyr?Pro?Ser?Pro?Gln?His?Met

725?????????????????730?????????????????735

Val?Gln?Thr?Asn?His?Tyr?Gln?Val?Ser?Gly?Tyr?Pro?Gly?Ser?His?Gly

740?????????????????745?????????????????750

Ile?Thr?Ala?Met?Ala?Gly?Ser?Ile?Tyr?Pro?Gly?Gln?Ala?Ser?Leu?Leu

755?????????????????760?????????????????765

Asp?Gln?Thr?Asp?Ser?Trp?Asn?His?Arg?Pro?Gln?Glu?Ile?Ala?Met?Trp

770?????????????????775?????????????????780

Gln?Pro?Asn?Val?Glu?Asp?Ser?Thr?Val?Leu?Asp?Leu?Arg?Gly?Ile?Gly

785?????????????????790?????????????????795?????????????????800

Gln?Val?Leu?Pro?Thr?His?Leu?Met?Glu?Glu?Arg?Leu?Ile?Arg?Gln?Gln

805?????????????????810?????????????????815

Gln?Glu?Met?Glu?Glu?Asp?Gln?Arg?Trp?Leu?Glu?Lys?Glu?Glu?Arg?Phe

820?????????????????825?????????????????830

Leu?Lys?Pro?Asp?Val?Arg?Leu?Ser?Arg?Gly?Ser?Ile?Asp?Arg?Glu?Asp

835?????????????????840?????????????????845

Gly?Ser?Leu?Gln?Gly?Pro?Ile?Gly?Asn?Gln?His?Ile?Tyr?Gln?Pro?Val

850?????????????????855?????????????????860

Gly?Lys?Pro?Asp?Pro?Ala?Ala?Pro?Pro?Lys?Lys?Pro?Pro?Arg?Pro?Gly

865?????????????????870?????????????????875?????????????????880

Ala?Pro?Gly?His?Leu?Gly?Ser?Leu?Ala?Ser?Leu?Ser?Ser?Pro?Ala?Asp

885?????????????????890?????????????????895

Ser?Tyr?Asn?Glu?Gly?Val?Lys?Leu?Gln?Pro?Gln?Glu?Ile?Ser?Pro?Pro

900?????????????????905?????????????????910

Pro?Thr?Ala?Asn?Leu?Asp?Arg?Ser?Asn?Asp?Lys?Val?Tyr?Glu?Asn?Val

915?????????????????920?????????????????925

Thr?Gly?Leu?Val?Lys?Ala?Val?Ile?Glu?Met?Ser?Ser?Lys?Ile?Gln?Pro

930?????????????????935?????????????????940

Ala?Pro?Pro?Glu?Glu?Tyr?Val?Pro?Met?Val?Lys?Glu?Val?Gly?Leu?Ala

945?????????????????950?????????????????955?????????????????960

Leu?ATg?Thr?Leu?Leu?Ala?Thr?Val?Asp?Glu?Thr?Ile?Pro?Leu?Leu?Pro

965?????????????????970?????????????????975

Ala?Ser?Thr?His?Arg?Glu?Ile?Glu?Met?Ala?Gln?Lys?Leu?Leu?Asn?Ser

980?????????????????985?????????????????990

Asp?Leu?Gly?Glu?Leu?Ile?Asn?Lys?Met?Lys?Leu?Ala?Gln?Gln?Tyr?Val

995?????????????????1000????????????????1005

Met?Thr?Ser?Leu?Gln?Gln?Glu?Tyr?Lys?Lys?Gln?Met?Leu?Thr?Ala

1010????????????????1015????????????????1020

Ala?His?Ala?Leu?Ala?Val?Asp?Ala?Lys?Asn?Leu?Leu?Asp?Val?Ile

1025????????????????1030????????????????1035

Asp?Gln?Ala?Arg?Leu?Lys?Met?Leu?Gly?Gln?Thr?Arg?Pro?His

1040????????????????1045????????????????1050

<210>4

<211>366

<212>PRT

＜213〉(wild-type FAK 694-1052 amino acids comprises initial sub-M to people FRNK; Do not comprise V5 epi-position and His mark)

<400>4

Met?Asp?Tyr?Pro?Tyr?Asp?Val?Glu?Ser?Arg?Arg?Gln?Ala?Thr?Val?Ser

1???????????????5???????????????????10??????????????????15

Trp?Asp?Ser?Gly?Gly?Ser?Asp?Glu?Ala?Pro?Pro?Lys?Pro?Ser?Arg?Pro

20??????????????????25??????????????????30

Gly?Tyr?Pro?Ser?Pro?Arg?Ser?Ser?Glu?Gly?Phe?Tyr?Pro?Ser?Pro?Gln

35??????????????????40??????????????????45

His?Met?Val?Gln?Thr?Asn?His?Tyr?Gln?Val?Ser?Gly?Tyr?Pro?Gly?Ser

50??????????????????55??????????????????60

His?Gly?Ile?Thr?Ala?Met?Ala?Gly?Ser?lle?Tyr?Pro?Gly?Gln?Ala?Ser

65??????????????????70??????????????????75??????????????????80

Leu?Leu?Asp?Gln?Thr?Asp?Ser?Trp?Asn?His?Arg?Pro?Gln?Glu?Ile?Ala

85??????????????????90??????????????????95

Met?Trp?Gln?Pro?Asn?Val?Glu?Asp?Ser?Thr?Val?Leu?Asp?Leu?Arg?Gly

100?????????????????105?????????????????110

Ile?Gly?Gln?Val?Leu?Pro?Thr?His?Leu?Met?Glu?Glu?Arg?Leu?Ile?Arg

115?????????????????120?????????????????125

Gln?Gln?Gln?Glu?Met?Glu?Glu?Asp?Gln?Arg?Trp?Leu?Glu?Lys?Glu?Glu

130?????????????????135?????????????????140

Arg?Phe?Leu?Lys?Pro?Asp?Val?Arg?Leu?Ser?Arg?Gly?Ser?Ile?Asp?Arg

145?????????????????150?????????????????155?????????????????160

Glu?Asp?Gly?Ser?Leu?Gln?Gly?Pro?Ile?Gly?Asn?Gln?His?Ile?Tyr?Gln

165?????????????????170?????????????????175

Pro?Val?Gly?Lys?Pro?Asp?Pro?Ala?Ala?Pro?Pro?Lys?Lys?Pro?Pro?Arg

180?????????????????185?????????????????190

Pro?Gly?Ala?Pro?Gly?His?Leu?Gly?Ser?Leu?Ala?Ser?Leu?Ser?Ser?Pro

195?????????????????200?????????????????205

Ala?Asp?Ser?Tyr?Asn?Glu?Gly?Val?Lys?Leu?Gln?Pro?Gln?Glu?Ile?Ser

210?????????????????215?????????????????220

Pro?Pro?Pro?Thr?Ala?Asn?Leu?Asp?Arg?Ser?Asn?Asp?Lys?Val?Tyr?Glu

225?????????????????230?????????????????235?????????????????240

Asn?Val?Thr?Gly?Leu?Val?Lys?Ala?Val?Ile?Glu?Met?Ser?Ser?Lys?Ile

245?????????????????250?????????????????255

Gln?Pro?Ala?Pro?Pro?Glu?Glu?Tyr?Val?Pro?Met?Val?Lys?Glu?Val?Gly

260?????????????????265?????????????????270

Leu?Ala?Leu?Arg?Thr?Leu?Leu?Ala?Thr?Val?Asp?Glu?Thr?Ile?Pro?Leu

275?????????????????280?????????????????285

Leu?Pro?Ala?Ser?Thr?His?Arg?Glu?Ile?Glu?Met?Ala?Gln?Lys?Leu?Leu

290?????????????????295?????????????????300

Asn?Ser?Asp?Leu?Gly?Glu?Leu?Ile?Asn?Lys?Met?Lys?Leu?Ala?Gln?Gln

305?????????????????310?????????????????315?????????????????320

Tyr?Val?Met?Thr?Ser?Leu?Gln?Gln?Glu?Tyr?Lys?Lys?Gln?Met?Leu?Thr

325?????????????????330?????????????????335

Ala?Ala?His?Ala?Leu?Ala?Val?Asp?Ala?Lys?Asn?Leu?Leu?Asp?Val?Ile

340?????????????????345?????????????????350

Asp?Gln?Ala?Arg?Leu?Lys?Met?Leu?Gly?Gln?Thr?Arg?Pro?His

355?????????????????360?????????????????365

<210>5

<211>3334

<212>DNA

＜213〉human FAK (pGene/FAKwt/V5-His)

<400>5

tgcagtatct?gtatttttgc?tagcagtaat?actaacggtt?ctttttttct?cttcacaggc????60

caccaagctt?ggtaccgagc?tcggatccat?ggcagctgct?taccttgacc?ccaacttgaa????120

tcacacacca?aattcgagta?ctaagactca?cctgggtact?ggtatggaac?gttctcctgg????180

tgcaatggag?cgagtattaa?aggtctttca?ttattttgaa?agcaatagtg?agccaaccac????240

ctgggccagt?attatcaggc?atggagatgc?tactgatgtc?aggggcatca?ttcagaagat????300

agtggacagt?cacaaagtaa?agcatgtggc?ctgctatgga?ttccgcctca?gtcacctgcg????360

gtcagaggag?gttcactggc?ttcacgtgga?tatgggcgtc?tccagtgtga?gggagaagta????420

tgagcttgct?cacccaccag?aggagtggaa?atatgaattg?agaattcgtt?atttgccaaa????480

aggatttcta?aaccagttta?ctgaagataa?gccaactttg?aatttcttct?atcaacaggt????540

gaagagcgat?tatatgttag?agatagccga?tcaagtggac?caggaaattg?ctttgaagtt????600

gggttgtcta?gaaatacggc?gatcatactg?ggagatgcgg?ggcaatgcac?tagaaaagaa????660

gtctaactat?gaagtattag?aaaaagatgt?tggtttaaag?cgattttttc?ctaagagttt????720

actggattct?gtcaaggcca?aaacactaag?aaaactgatc?caacaaacat?ttagacaatt????780

tgccaacctt?aatagagaag?aaagtattct?gaaattcttt?gagatcctgt?ctccagtcta????840

cagatttgat?aaggaatgct?tcaagtgtgc?tcttggttca?agctggatta?tttcagtgga????900

actggcaatc?ggcccagaag?aaggaatcag?ttacctaacg?gacaagggct?gcaatcccac????960

acatcttgct?gacttcactc?aagtgcaaac?cattcagtat?tcaaacagtg?aagacaagga????1020

cagaaaagga?atgctacaac?taaaaatagc?aggtgcaccc?gagcctctga?cagtgacggc????1080

accatcccta?accattgcgg?agaatatggc?tgacctaata?gatgggtact?gccggctggt????1140

gaatggaacc?tcgcagtcat?ttatcatcag?acctcagaaa?gaaggtgaac?gggctttgcc????1200

atcaatacca?aagttggcca?acagcgaaaa?gcaaggcatg?cggacacacg?ccgtctctgt????1260

gtcagaaaca?gatgattatg?ctgagattat?agatgaagaa?gatacttaca?ccatgccctc????1320

aaccagggat?tatgagattc?aaagagaaag?aatagaactt?ggacgatgta?ttggagaagg????1380

ccaatttgga?gatgtacatc?aaggcattta?tatgagtcca?gagaatccag?ctttggcggt????1440

tgcaattaaa?acatgtaaaa?actgtacttc?ggacagcgtg?agagagaaat?ttcttcaaga????1500

agccttaaca?atgcgtcagt?ttgaccatcc?tcatattgtg?aagctgattg?gagtcatcac????1560

agagaatcct?gtctggataa?tcatggagct?gtgcacactt?ggagagctga?ggtcattttt????1620

gcaagtaagg?aaatacagtt?tggatctagc?atctttgatc?ctgtatgcct?atcagcttag????1680

tacagctctt?gcatatctag?agagcaaaag?atttgtacac?agggacattg?ctgctcggaa????1740

tgttctggtg?tcctcaaatg?attgtgtaaa?attaggagac?tttggattat?cccgatatat????1800

ggaagatagt?acttactaca?aagcttccaa?aggaaaattg?cctattaaat?ggatggctcc????1860

agagtcaatc?aattttcgac?gttttacctc?agctagtgac?gtatggatgt?ttggtgtgtg????1920

tatgtgggag?atactgatgc?atggtgtgaa?gccttttcaa?ggagtgaaga?acaatgatgt????1980

aatcggtcga?attgaaaatg?gggaaagatt?accaatgcct?ccaaattgtc?ctcctaccct????2040

ctacagcctt?atgacgaaat?gctgggccta?tgaccccagc?aggcggccca?ggtttactga????2100

acttaaagct?cagctcagca?caatcctgga?ggaagagaag?gctcagcaag?aagagcgcat????2160

gaggatggag?tccagaagac?aggccacagt?gtcctgggac?tccggagggt?ctgatgaagc????2220

accgcccaag?cccagcagac?cgggttatcc?cagtccgagg?tccagcgaag?gattttatcc????2280

cagcccacag?cacatggtac?aaaccaatca?ttaccaggtt?tctggctacc?ctggttcaca????2340

tggaatcaca?gccatggctg?gcagcatcta?tccaggtcag?gcatctcttt?tggaccaaac????2400

agattcatgg?aatcatagac?ctcaggagat?agcaatgtgg?cagcccaatg?tggaggactc????2460

tacagtattg?gacctgcgag?ggattgggca?agtgttgcca?acccatctga?tggaagagcg????2520

tctaatccga?cagcaacagg?aaatggaaga?agatcagcgc?tggctggaaa?aagaggaaag????2580

atttctgaaa?cctgatgtga?gactctctcg?aggcagtatt?gacagggagg?atggaagtct????2640

tcagggtccg?attggaaacc?aacatatata?tcagcctgtg?ggtaaaccag?atcctgcagc????2700

tccaccaaag?aaaccgcctc?gccctggagc?tcccggtcat?ctgggaagcc?ttgccagcct????2760

cagcagccct?gctgacagct?acaacgaggg?tgtcaagctt?cagccccagg?aaatcagccc????2820

ccctcctact?gccaacctgg?accggtcgaa?tgataaggtg?tacgagaatg?tgacgggcct????2880

ggtgaaagct?gtcatcgaga?tgtccagtaa?aatccagcca?gccccaccag?aggagtatgt????2940

ccctatggtg?aaggaagtcg?gcttggccct?gaggacatta?ttggccactg?tggatgagac????3000

cattcccctc?ctaccagcca?gcacccaccg?agagattgag?atggcacaga?agctattgaa????3060

ctctgacctg?ggtgagctca?tcaacaagat?gaaactggcc?cagcagtatg?tcatgaccag????3120

cctccagcaa?gagtacaaaa?agcaaatgct?gactgctgct?cacgccctgg?ctgtggatgc????3180

caaaaactta?ctcgatgtca?ttgaccaagc?aagactgaaa?atgcttgggc?agacgagacc????3240

acactccctg?gggcccttcg?aaggtaagcc?tatccctaac?cctctcctcg?gtctcgattc????3300

tacgcgtacc?ggtcatcatc?accatcacca?ttga????????????????????????????????3334

<210>6

<211>3308

<212>DNA

＜213〉human FAK Y397F mutant (pGene/FAKY397F/V5-His)

<400>6

taatactaac?ggttcttttt?ttctcttcac?aggccaccaa?gcttggtacc?gagctcggat????60

ccatggcagc?tgcttacctt?gaccccaact?tgaatcacac?accaaattcg?agtactaaga????120

ctcacctggg?tactggtatg?gaacgttctc?ctggtgcaat?ggagcgagta?ttaaaggtct????180

ttcattattt?tgaaagcaat?agtgagccaa?ccacctgggc?cagtattatc?aggcatggag????240

atgctactga?tgtcaggggc?atcattcaga?agatagtgga?cagtcacaaa?gtaaagcatg????300

tggcctgcta?tggattccgc?ctcagtcacc?tgcggtcaga?ggaggttcac?tggcttcacg????360

tggatatggg?cgtctccagt?gtgagggaga?agtatgagct?tgctcaccca?ccagaggagt????420

ggaaatatga?attgagaatt?cgttatttgc?caaaaggatt?tctaaaccag?tttactgaag????480

ataagccaac?tttgaatttc?ttctatcaac?aggtgaagag?cgattatatg?ttagagatag????540

ccgatcaagt?ggaccaggaa?attgctttga?agttgggttg?tctagaaata?cggcgatcat????600

actgggagat?gcggggcaat?gcactagaaa?agaagtctaa?ctatgaagta?ttagaaaaag????660

atgttggttt?aaagcgattt?tttcctaaga?gtttactgga?ttctgtcaag?gccaaaacac????720

taagaaaact?gatccaacaa?acatttagac?aatttgccaa?ccttaataga?gaagaaagta????780

ttctgaaatt?ctttgagatc?ctgtctccag?tctacagatt?tgataaggaa?tgcttcaagt????840

gtgctcttgg?ttcaagctgg?attatttcag?tggaactggc?aatcggccca?gaagaaggaa????900

tcagttacct?aacggacaag?ggctgcaatc?ccacacatct?tgctgacttc?actcaagtgc????960

aaaccattca?gtattcaaac?agtgaagaca?aggacagaaa?aggaatgcta?caactaaaaa????1020

tagcaggtgc?acccgagcct?ctgacagtga?cggcaccatc?cctaaccatt?gcggagaata????1080

tggctgacct?aatagatggg?tactgccggc?tggtgaatgg?aacctcgcag?tcatttatca????1140

tcagacctca?gaaagaaggt?gaacgggctt?tgccatcaat?accaaagttg?gccaacagcg????1200

aaaagcaagg?catgcggaca?cacgccgtct?ctgtgtcaga?aacagatgat?tttgctgaga????1260

ttatagatga?agaagatact?tacaccatgc?cctcaaccag?ggattatgag?attcaaagag????1320

aaagaataga?acttggacga?tgtattggag?aaggccaatt?tggagatgta?catcaaggca????1380

tttatatgag?tccagagaat?ccagctttgg?cggttgcaat?taaaacatgt?aaaaactgta????1440

cttcggacag?cgtgagagag?aaatttcttc?aagaagcctt?aacaatgcgt?cagtttgacc????1500

atcctcatat?tgtgaagctg?attggagtca?tcacagagaa?tcctgtctgg?ataatcatgg????1560

agctgtgcac?acttggagag?ctgaggtcat?ttttgcaagt?aaggaaatac?agtttggatc????1620

tagcatcttt?gatcctgtat?gcctatcagc?ttagtacagc?tcttgcatat?ctagagagca????1680

aaagatttgt?acacagggac?attgctgctc?ggaatgttct?ggtgtcctca?aatgattgtg????1740

taaaattagg?agactttgga?ttatcccgat?atatggaaga?tagtacttac?tacaaagctt????1800

ccaaaggaaa?attgcctatt?aaatggatgg?ctccagagtc?aatcaatttt?cgacgtttta????1860

cctcagctag?tgacgtatgg?atgtttggtg?tgtgtatgtg?ggagatactg?atgcatggtg????1920

tgaagccttt?tcaaggagtg?aagaacaatg?atgtaatcgg?tcgaattgaa?aatggggaaa????1980

gattaccaat?gcctccaaat?tgtcctccta?ccctctacag?ccttatgacg?aaatgctggg????2040

cctatgaccc?cagcaggcgg?cccaggttta?ctgaacttaa?agctcagctc?agcacaatcc????2100

tggaggaaga?gaaggctcag?caagaagagc?gcatgaggat?ggagtccaga?agacaggcca????2160

cagtgtcctg?ggactccgga?gggtctgatg?aagcaccgcc?caagcccagc?agaccgggtt????2220

atcccagtcc?gaggtccagc?gaaggatttt?atcccagccc?acagcacatg?gtacaaacca????2280

atcattacca?ggtttctggc?taccctggtt?cacatggaat?cacagccatg?gctggcagca????2340

tctatccagg?tcaggcatct?cttttggacc?aaacagattc?atggaatcat?agacctcagg????2400

agatagcaat?gtggcagccc?aatgtggagg?actctacagt?attggacctg?cgagggattg????2460

ggcaagtgtt?gccaacccat?ctgatggaag?agcgtctaat?ccgacagcaa?caggaaatgg????2520

aagaagatca?gcgctggctg?gaaaaagagg?aaagatttct?gaaacctgat?gtgagactct????2580

ctcgaggcag?tattgacagg?gaggatggaa?gtcttcaggg?tccgattgga?aaccaacata????2640

tatatcagcc?tgtgggtaaa?ccagatcctg?cagctccacc?aaagaaaccg?cctcgccctg????2700

gagctcccgg?tcatctggga?agccttgcca?gcctcagcag?ccctgctgac?agctacaacg????2760

agggtgtcaa?gcttcagccc?caggaaatca?gcccccctcc?tactgccaac?ctggaccggt????2820

cgaatgataa?ggtgtacgag?aatgtgacgg?gcctggtgaa?agctgtcatc?gagatgtcca????2880

gtaaaatcca?gccagcccca?ccagaggagt?atgtccctat?ggtgaaggaa?gtcggcttgg????2940

ccctgaggac?attattggcc?actgtggatg?agaccattcc?cctcctacca?gccagcaccc????3000

accgagagat?tgagatggca?cagaagctat?tgaactctga?cctgggtgag?ctcatcaaca????3060

agatgaaact?ggcccagcag?tatgtcatga?ccagcctcca?gcaagagtac?aaaaagcaaa????3120

tgctgactgc?tgctcacgcc?ctggctgtgg?atgccaaaaa?cttactcgat?gtcattgacc????3180

aagcaagact?gaaaatgctt?gggcagacga?gaccacactc?cctggggccc?ttcgaaggta????3240

agcctatccc?taaccctctc?ctcggtctcg?attctacgcg?taccggtcat?catcaccatc????3300

accattga?????????????????????????????????????????????????????????????3308

<210>7

<211>3323

<212>DNA

＜213〉human FAK K454R mutant (pGene/FAKK454R/V5His)

<400>7

tatttttgct?agcagtaata?ctaacggttc?tttttttctc?ttcacaggcc?accaagcttg????60

gtaccgagct?cggatccatg?gcagctgctt?accttgaccc?caacttgaat?cacacaccaa????120

attcgagtac?taagactcac?ctgggtactg?gtatggaacg?ttctcctggt?gcaatggagc????180

gagtattaaa?ggtctttcat?tattttgaaa?gcaatagtga?gccaaccacc?tgggccagta????240

ttatcaggca?tggagatgct?actgatgtca?ggggcatcat?tcagaagata?gtggacagtc????300

acaaagtaaa?gcatgtggcc?tgctatggat?tccgcctcag?tcacctgcgg?tcagaggagg????360

ttcactggct?tcacgtggat?atgggcgtct?ccagtgtgag?ggagaagtat?gagcttgctc????420

acccaccaga?ggagtggaaa?tatgaattga?gaattcgtta?tttgccaaaa?ggatttctaa????480

accagtttac?tgaagataag?ccaactttga?atttcttcta?tcaacaggtg?aagagcgatt????540

atatgttaga?gatagccgat?caagtggacc?aggaaattgc?tttgaagttg?ggttgtctag????600

aaatacggcg?atcatactgg?gagatgcggg?gcaatgcact?agaaaagaag?tctaactatg????660

aagtattaga?aaaagatgtt?ggtttaaagc?gattttttcc?taagagttta?ctggattctg????720

tcaaggccaa?aacactaaga?aaactgatcc?aacaaacatt?tagacaattt?gccaacctta????780

atagagaaga?aagtattctg?aaattctttg?agatcctgtc?tccagtctac?agatttgata????840

aggaatgctt?caagtgtgct?cttggttcaa?gctggattat?ttcagtggaa?ctggcaatcg????900

gcccagaaga?aggaatcagt?tacctaacgg?acaagggctg?caatcccaca?catcttgctg????960

acttcactca?agtgcaaacc?attcagtatt?caaacagtga?agacaaggac?agaaaaggaa????1020

tgctacaact?aaaaatagca?ggtgcacccg?agcctctgac?agtgacggca?ccatccctaa????1080

ccattgcgga?gaatatggct?gacctaatag?atgggtactg?ccggctggtg?aatggaacct????1140

cgcagtcatt?tatcatcaga?cctcagaaag?aaggtgaacg?ggctttgcca?tcaataccaa????1200

agttggccaa?cagcgaaaag?caaggcatgc?ggacacacgc?cgtctctgtg?tcagaaacag????1260

atgattatgc?tgagattata?gatgaagaag?atacttacac?catgccctca?accagggatt????1320

atgagattca?aagagaaaga?atagaacttg?gacgatgtat?tggagaaggc?caatttggag????1380

atgtacatca?aggcatttat?atgagtccag?agaatccagc?tttggcggtt?gcaattagaa????1440

catgtaaaaa?ctgtacttcg?gacagcgtga?gagagaaatt?tcttcaagaa?gccttaacaa????1500

tgcgtcagtt?tgaccatcct?catattgtga?agctgattgg?agtcatcaca?gagaatcctg????1560

tctggataat?catggagctg?tgcacacttg?gagagctgag?gtcatttttg?caagtaagga????1620

aatacagttt?ggatctagca?tctttgatcc?tgtatgccta?tcagcttagt?acagctcttg????1680

catatctaga?gagcaaaaga?tttgtacaca?gggacattgc?tgctcggaat?gttctggtgt????1740

cctcaaatga?ttgtgtaaaa?ttaggagact?ttggattatc?ccgatatatg?gaagatagta????1800

cttactacaa?agcttccaaa?ggaaaattgc?ctattaaatg?gatggctcca?gagtcaatca????1860

attttcgacg?ttttacctca?gctagtgacg?tatggatgtt?tggtgtgtgt?atgtgggaga????1920

tactgatgca?tggtgtgaag?ccttttcaag?gagtgaagaa?caatgatgta?atcggtcgaa????1980

ttgaaaatgg?ggaaagatta?ccaatgcctc?caaattgtcc?tcctaccctc?tacagcctta????2040

tgacgaaatg?ctgggcctat?gaccccagca?ggcggcccag?gtttactgaa?cttaaagctc????2100

agctcagcac?aatcctggag?gaagagaagg?ctcagcaaga?agagcgcatg?aggatggagt????2160

ccagaagaca?ggccacagtg?tcctgggact?ccggagggtc?tgatgaagca?ccgcccaagc????2220

ccagcagacc?gggttatccc?agtccgaggt?ccagcgaagg?attttatccc?agcccacagc????2280

acatggtaca?aaccaatcat?taccaggttt?ctggctaccc?tggttcacat?ggaatcacag????2340

ccatggctgg?cagcatctat?ccaggtcagg?catctctttt?ggaccaaaca?gattcatgga????2400

atcatagacc?tcaggagata?gcaatgtggc?agcccaatgt?ggaggactct?acagtattgg????2460

acctgcgagg?gattgggcaa?gtgttgccaa?cccatctgat?ggaagagcgt?ctaatccgac????2520

agcaacagga?aatggaagaa?gatcagcgct?ggctggaaaa?agaggaaaga?tttctgaaac????2580

ctgatgtgag?actctctcga?ggcagtattg?acagggagga?tggaagtctt?cagggtccga????2640

ttggaaacca?acatatatat?cagcctgtgg?gtaaaccaga?tcctgcagct?ccaccaaaga????2700

aaccgcctcg?ccctggagct?cccggtcatc?tgggaagcct?tgccagcctc?agcagccctg????2760

ctgacagcta?caacgagggt?gtcaagcttc?agccccagga?aatcagcccc?cctcctactg????2820

ccaacctgga?ccggtcgaat?gataaggtgt?acgagaatgt?gacgggcctg?gtgaaagctg????2880

tcatcgagat?gtccagtaaa?atccagccag?ccccaccaga?ggagtatgtc?cctatggtga????2940

aggaagtcgg?cttggccctg?aggacattat?tggccactgt?ggatgagacc?attcccctcc????3000

taccagccag?cacccaccga?gagattgaga?tggcacagaa?gctattgaac?tctgacctgg????3060

gtgagctcat?caacaagatg?aaactggccc?agcagtatgt?catgaccagc?ctccagcaag????3120

agtacaaaaa?gcaaatgctg?actgctgctc?acgccctggc?tgtggatgcc?aaaaacttac????3180

tcgatgtcat?tgaccaagca?agactgaaaa?tgcttgggca?gacgagacca?cactccctgg????3240

ggcccttcga?aggtaagcct?atccctaacc?ctctcctcgg?tctcgattct?acgcgtaccg????3300

gtcatcatca?ccatcaccat?tga????????????????????????????????????????????3323

<210>8

<211>1234

<212>DNA

＜213〉be called the human FAK mutant (pGene/FRNK/V5-His) of FRNK

<400>8

cggttctttt?tttctcttca?caggccacca?agcttggtac?cgccaccatg?gactacccct????60

atgatgtgga?gtccagaaga?caggccacag?tgtcctggga?ctccggaggg?tctgatgaag????120

caccgcccaa?gcccagcaga?ccgggttatc?ccagtccgag?gtccagcgaa?ggattttatc????180

ccagcccaca?gcacatggta?caaaccaatc?attaccaggt?ttctggctac?cctggttcac????240

atggaatcac?agccatggct?ggcagcatct?atccaggtca?ggcatctctt?ttggaccaaa????300

cagattcatg?gaatcataga?cctcaggaga?tagcaatgtg?gcagcccaat?gtggaggact????360

ctacagtatt?ggacctgcga?gggattgggc?aagtgttgcc?aacccatctg?atggaagagc????420

gtctaatccg?acagcaacag?gaaatggaag?aagatcagcg?ctggctggaa?aaagaggaaa????480

gatttctgaa?acctgatgtg?agactctctc?gaggcagtat?tgacagggag?gatggaagtc????540

ttcagggtcc?gattggaaac?caacatatat?atcagcctgt?gggtaaacca?gatcctgcag????600

ctccaccaaa?gaaaccgcct?cgccctggag?ctcccggtca?tctgggaagc?cttgccagcc????660

tcagcagccc?tgctgacagc?tacaacgagg?gtgtcaagct?tcagccccag?gaaatcagcc????720

cccctcctac?tgccaacctg?gaccggtcga?atgataaggt?gtacgagaat?gtgacgggcc????780

tggtgaaagc?tgtcatcgag?atgtccagta?aaatccagcc?agccccacca?gaggagtatg????840

tccctatggt?gaaggaagtc?ggcttggccc?tgaggacatt?attggccact?gtggatgaga????900

ccattcccct?cctaccagcc?agcacccacc?gagagattga?gatggcacag?aagctattga????960

actctgacct?gggtgagctc?atcaacaaga?tgaaactggc?ccagcagtat?gtcatgacca????1020

gcctccagca?agagtacaaa?aagcaaatgc?tgactgctgc?tcacgccctg?gctgtggatg????1080

ccaaaaactt?actcgatgtc?attgaccaag?caagactgaa?aatgcttggg?cagacgagac????1140

cacactccct?gggcccttcg?aaggtaagcc?tatccctaac?cctctcctcg?gtctcgattc????1200

tacgcgtacc?ggtcatcatc?accatcacca??ttga???????????????????????????????1234

<210>9

<211>27

<212>DNA

＜213〉FAK5 ' Bam primer

<400>9

ggatccatgg?cagctgctta?ccttgac????????????????????????????????????????27

<210>10

<211>36

<212>DNA

＜213〉FAK3 ' Bam primer

<400>10

ggatcctcac?tcactcagtg?tggtctcgtc?tgccca??????????????????????????????36

Claims (14)

1. identify the method for the cell-activity inhibitor of focal adhesion kinase (FAK), comprising:

(a) add inductor to induce the expression of gene of coding FAK in mammalian cell, wherein said mammalian cell is by described stable gene transfection, and described gene is expressed when described inductor exists;

(b) add test compound;

(c) utilize the FAK trapping agent to catch expressed FAK; And

(d) phosphorylation of the described FAK of detection.

2. the described method of claim 1 wherein is coated on described mammalian cell on first solid phase, and wherein contacts and detect the existence of described antibody, detect the phosphorylation of FAK with anti--phosphoric acid-tyrosine antibody by the FAK with phosphorylation.

3. the described method of claim 1, wherein said FAK trapping agent comprises the antibody of one or more types.

4. the described method of claim 3, the antibody of wherein said one or more types comprises anti-phosphoric acid-tyrosine antibody.

5. the described method of claim 1, wherein said phosphorylation and anti-phosphoric acid-tyrosine antibody are to the ratio that is combined into of the FAK that caught.

6. the described method of claim 2, wherein said cell and first solid phase is natural adheres to, and wherein said cell was used the lysis buffer cracking before catching the FAK that expresses.

7. the described method of claim 1, wherein said inductor are the agonists of the expression of gene of coding FAK.

8. the described method of claim 1, wherein said test compound suppresses the kinases dependency phosphorylation of FAK.

9. measure the Cytotoxic method of test compound, comprising:

(a) with the stable gene transfection mammalian cell of coding FAK, wherein said gene is expressed when inductor exists;

(b) add the genetic expression that inductor is induced described coding FAK;

(c) add test compound;

(d) cytotoxicity is added to described cell; And,

(e) cytotoxicity of the described test compound of detection.

10. with the mammalian cell of nucleic acid molecule stable transfection of reorganization, the nucleic acid molecule encoding of this reorganization comprises and is selected from SEQ ID NO:1, the albumen of 2,3 and 4 sequence, and wherein said proteic expression needs the existence of inductor.

11. with the nucleic acid stability mammalian cells transfected of reorganization, its inducibility is expressed FAK albumen.

12. the described mammalian cell of claim 11, wherein said nucleic acid encoding is selected from following albumen: human FAK splice variant, human FAK catalytic domain, rat FAK, mouse FAK and chicken FAK.

13. with the mammalian cell of nucleic acid molecule stable transfection of reorganization, it comprises and is selected from SEQ IDNO:5,6,7 and 8 polynucleotide, and the expression of wherein said polynucleotide needs the existence of inductor.

14. identify the method for the cell-activity inhibitor of focal adhesion kinase (FAK), comprise the following steps:

(a) with the mammalian cell bag of a group homogeneous by first solid phase, make cell adhesion on first solid phase, the be encoded stable gene transfection of FAK of wherein said cell, and wherein said gene is expressed when inductor exists;

(b) add the genetic expression that inductor is induced described coding FAK;

(c) add test compound;

(d) the described adherent cell of dissolving discharges cell lysate;

(e) use FAK trapping agent bag by second solid phase, the FAK trapping agent is attached on this second solid phase;

(f) described cell lysate is contacted with the FAK trapping agent that adheres to, thereby allow the FAK trapping agent catch FAK;

(g) make the FAK of seizure be exposed to anti-phosphoric acid-tyrosine antibody, and,

(h) detect the combining of FAK of anti-phosphoric acid-tyrosine antibody and seizure, wherein the phosphorylation amount of the anti-phosphoric acid-tyrosine antibody amount of the FAK of integrating capture and described FAK is proportional.

Images