CA2497434A1

CA2497434A1 - Inducible focal adhesion kinase cell assay

Info

Publication number: CA2497434A1
Application number: CA002497434A
Authority: CA
Inventors: Walter Gregory Roberts; Pamela Matthews Whalen; Ethan James Tekly Ung
Original assignee: Individual
Current assignee: Pfizer Products Inc
Priority date: 2002-09-19
Filing date: 2003-09-08
Publication date: 2004-04-01
Also published as: KR20050052663A; JP2006500022A; WO2004027018A2; EP1546372A2; AU2003259498A1; WO2004027018A3; NO20051869D0; NO20051869L; RU2005107701A; CN1681939A; EP1546372A4

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 (Y397). 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

INDUCIBLE FOCAL ADHESION KINASE CELL ASSAY
Background of the Invention This invention relates to methods and compositions for inducing the expression of the focal adhesion kinase (FAK) gene, which encodes a signaling protein involved in growth factor response and cell migration and is also implicated in disease. The invention is also directed to the identification of FAK inhibitors.
FAK is a cytoplasmic, non-receptor tyrosine kinase. FAK transducer signaling from a diverse group of stimuli (e.g. integrins, cytokines, chemokines, and growth factors) to control a variety of cellular pathways and processes including cell proliferation, migration, morphology, and cell survival. In addition to being expressed in most tissue types, FAK is found at elevated levels in most human cancers, particularly in highly invasive metastases. It has been shown that expression of the dominant-negative FAK-related nonkinase (FRNK) in human tumor cells results in rounded morphology of the cells, the irreversible loss of focal plaques, and subsequent cell death. In addition, the controlled expression of FRNK results in decreased tyrosine phosphorylation of FAK, suggesting that inhibition of FAK
phosphorylation may yield a therapeutic index for the treatment of human cancers.
Although the exact mechanisms leading to FAK activation are not well defined, it is believed that FAK enzyme activity resulting in phosphorylation at Y397 (tyrosine residue at position 397) is the critical step in integrin signal transduction (Guan, JL, Int. J.
Biochem.CeILBioI. 29: 1085-96, 1997). The transmembrane integrin receptors are important for linking the extracellular matrix (ECM) proteins with the cellular actin cytoskeleton and the nucleus to regulate cell morphology, tissue architecture, and attachment-induced gene expression. It is believed that colocalization of integrin receptors and FAK
to sites of focal adhesion leads to FAK phosphorylation at residue Y397, creating a SH2 docking site for Src-family tyrosine kinases. Src binding to phosphotyrosine FAKY397 leads to preferential phosphorylation of FAK at various downstream tyrosine residues including Y576, Y577, Y861, and Y925. Phosphorylation of FAK tyrosine residues (Y576/Y577) leads to increased FAK
kinase activity and signal transduction to regulate cytoskeletal reorganization, cell proliferation, cell survival, and cell migration.
Due to the multiplicity of kinases and substrates involved in the integrin signaling cascade, it is desirable to design assays specific for a particular kinase. An objective of the invention is to design and develop a FAK drug discovery pathway that tracks the biochemical mechanism of FAK. A number of exogenous stimuli can lead to FAK
phosphorylation such as S
(1 ) integrin binding to ECM ligands (e.g. Integrin ~i1 to Fibronectin); (2) cytokine or chemokine stimulation (e.g. Endothelin1/2, Bombesin, or PMA); (3) growth factor stimulation of tyrosine kinase receptors (e.g. PDGFBB); and (4) integrin antibody cross-finking (e.g.
Anti-~i1).

Conversely, the most feasible exogenous control leading to FAK inactivation is the detachment of cell-to-cell and cell-to-ECM contact (e.g. cell suspension).
SUMMARY OF THE INVENTION
The invention relates to a method for identifying cell-active inhibitors of focal adhesion kinase (FAK) comprising:
(a) adding an inducing agent to mammalian cells to induce the expression of a gene encoding FAK, wherein said mammalian cells are stably transfected with said gene, and said gene is expressed in the presence of said inducing agent;
(b) adding a test compound;
(c) capturing the expressed FAK using a FAK capture agent; and (d) detecting phosphorylation of said FAK.
An embodiment of the invention provides a method for measuring the cytotoxicity of a test compound comprising, stably transfecting mammalian cells with a gene encoding FAK, wherein said gene is expressed in the presence of an inducing agent; adding an inducing agent to induce the expression of said gene encoding FAK; adding a test compound; adding a cytotoxicity indicator to said cells; and, detecting the cytoxicity of the test compound. In certain embodiments, the cytoxicity of the test compound is determined by the colorimetric conversion of the cytotoxicity indicator, wherein the amount of converted cytotoxicity indicator is proportional to the number of living cells.
An embodiment of the present invention provides a mammalian cell stably transfected with a recombinant nucleic acid molecule, wherein said recombinant nucleic acid molecule is selected from the group consisting of SEQ ID No: 5, SEQ ID No: 6, SEQ ID No: 7 and SEQ ID
No: 8, and wherein expression of said sequences requires the presence of an inducing agent.
An embodiment of the invention provides a mammalian cell stably transfected with a recombinant nucleic acid molecule that encodes a protein comprising a sequence selected from the group consisting of SEQ ID NOS: 1, 2, 3 and 4, and wherein expression of said protein requires the presence of an inducing agent.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 shows a schematic representation of the detection of phosphorylated FAK
using a horseradish peroxidase-conjugated phosphotyrosine antibody (pY54"RP).
Figure 2 shows a schematic representation of the detection of phosphorylated FAK
using an unconjugated phosphotyrosine antibody (pY54) followed by a secondary mouse horseradish peroxidase antibody.
Figure 3 shows a schematic representation of a FAK inducible cell-based assay of the invention.

DETAILED DESCRIPTION
The invention is directed to an inducible cell-based assay for FAK. The cell-based assay exploits the biology of FAK and an inducible gene expression system to exogenously control FAK expression and FAK phosphorylation at the tyrosine residue at position 397 (Y397). By using a FAKY3s' phosphorylation-specific cell-based assay, rather than a general phosphotyrosine system, the identification of false-positive inhibitors is avoided. 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.
The inducible FAK cell-based assay of the present invention is advantageous in that it provides tight control over ectopic-basal level expression of FAK and rapid de-repression of FAK gene expression via an exogenous stimulant. The cell-based assay is flexible such that the final read-out is mechanistically relevant to FAK biology as measured for phosphotyrosine FAKY3s~, total FAK phosphotyrosine profile, FAK or mutant proteins, or some combination of a protein and phosphotyrosine. In addition, the present invention has been succesfully used to identify a number of FAK inhibitors. As used herein, the term "tight control"
refers to the controlled expression of the FAK gene that occurs in the presence of an exogenous stimulant.
In other words, the invention provides an inducible gene expression system for FAK, where the expression of FAK is induced in the presence of an appropriate inducible agent. The present invention provides a method for the inducible expression of FAK, wherein the regulated expression of the FAK gene does not adversely affect cell viability.
An embodiment of the invention is directed to a cell-based assay for the screening of FAK inhibitors. The cell-based assay exploits the biology of FAK and an inducible gene expression system to exogenously control FAK expression and FAK
phosphorylation at the tyrosine residue at position 397 (Y397). The cell-based assay is mechanistically relevant to FAK biology and measures changes in FAK phosphorylation. By using a FAKY3s~
phosphorylation-specific cell-based assay, rather than a general phosphotyrosine system, the identification of false-positive inhibitors is avoided. The cell-based assay of the present invention is flexible and can measure FAK phosphorylation, total FAK
phosphorylation, identify mutant FAK proteins and measure a combination of protein and phosphotyrosine.
An embodiment of the invention provides a method for identifying cell-active inhibitors of FAK comprising, stably transfecting mammalian cells with a gene encoding FAK, wherein said gene is expressed in the presence of an inducing agent; adding an inducing agent to induce the expression of said gene encoding FAK; adding a test compound;
capturing the expressed FAK using a FAK capture agent; exposing the captured FAK to an anti-phospho-tyrosine antibody; and, detecting the phosphorylation of said FAK. In some embodiments, the extent of phosphorylation of the FAK is determined by the binding of the anti-phospho-tyrosine antibody to the captured FAK, wherein the amount of anti-phospho-tyrosine antibody binding to the captured FAK is proportional to the amount of phosphorylation of said FAK.
In certain embodiments of the invention, the method for identifying cell-active inhibitors of FAK comprises an optional step of coating the mammalian cells on a first solid phase. The first solid phase is preferably a well of a first microtiter plate.
In other embodiments of the invention, the cells coated on the first solid phase are lysed with a lysis buffer, prior to the capture of the expressed FAK. The lysis buffer optionally comprises a solubilizing detergent. In certain embodiments, the FAK capture agent is coated on a second solid phase, which is preferably a well of a second microtiter plate.
In certain embodiments, the test compound inhibits the phosphorylation of FAK
at Y397.
An embodiment of the invention provides a method for measuring the cytotoxicity of a test compound comprising, stably transfecting mammalian cells with a gene encoding FAK, wherein said gene is expressed in the presence of an inducing agent; adding an inducing agent to induce the expression of said gene encoding FAK; adding a test compound; adding a cytotoxicity indicator to said cells; and, detecting the cytoxicity of the test compound. In certain embodiments, the cytoxicity of the test compound is determined by the colorimetric conversion of the cytotoxicity indicator, wherein the amount of converted cytotoxicity indicator is proportional to the number of living cells.
In certain embodiments of the invention, the method for measuring the cytotoxicity of a test compound comprises an optional step of coating the mammalian cells on a solid phase.
The solid phase is preferably a well of a first microtiter plate.
An embodiment of the invention provides a method for identifying cell-active inhibitors of focal adhesion kinase (FAK) comprising, coating a first solid phase with a homogeneous population of mammalian cells so that the cells adhere to the first solid phase, wherein said cells are stably transfected with a gene encoding FAK, and wherein said gene is expressed in the presence of an inducing agent; adding an inducing agent to induce the expression of said gene encoding FAK; adding a test compound; solubilizing the adhering cells to release the cell lysate; coating a second solid phase with 'a FAK capture agent so that the FAK capture agent adheres to the second solid phase; exposing the cell lysate to the adhered FAK
capture agent so that the FAK capture agent captures FAK; exposing the captured FAK to an anti-phospho-tyrosine antibody; and, measuring binding of the anti-phospho-tyrosine antibody to the captured FAK, wherein the amount of anti-phospho-tyrosine antibody binding to the captured FAK is proportional to the amount of phosphorylation of said FAK.
Another embodiment of the present invention provides a mammalian cell stably transfected with a recombinant nucleic acid molecule, wherein said recombinant nucleic acid molecule is selected from the group consisting of SEQ ID No: 5, SEQ ID No: 6, SEQ ID No: 7 and SEQ ID No: 8, and wherein expression of said sequences requires the presence of an inducing agent.
An embodiment of the invention provides a mammalian cell stably transfected with a recombinant nucleic acid molecule, that encodes a protein comprising a sequence selected from the group consisting of SEQ ID NOS: 1, 2, 3 and 4, and wherein expression of said protein requires the presence of an inducing agent.
FAK is also known as the Protein-Tyrosine Kinase 2, PTK2. Any active FAK
variant can be employed in the above assay. Inactive mutants can also be used in the assay for various control purposes. Additional variants of FAK that can be employed in the above described assay include, wild type (WT) human FAK at 153012 with 1052 amino acids (SEG2 ID N0:1 ); splice variants of FAK such as described in Andre, E. & Becker-Andre, M., Expression of an N-terminally truncated form of human focal adhesion kinase in brain.
Biochem. Biophys. Res. Commun. 190: 140-147, 1993 (describing an 879 amino acid variant at AAA35819, a 554 amino acid variant at PC1226 and a 431 amino acid variant at PC1227);
the 570 catalytic domain of FAK at XP 050337; mouse FAK at NP 032008.1 with amino acid identity (97%) to human FAK; mouse FAK carboxyl truncated variant at AAH30180.1 with 878/904 amino acid identity (97%) to amino acids 1-903 of human FAK; rat FAK at NP 037213.1 with 1020/1055 amino acid identity (96%) human FAK; FAK
variant at JC5494 with 1017!1055 amino acid identity (96%) to human FAK; chicken FAK at with 988/1054 amino acid identity (93%) to human FAK; chicken FAK variant at A45388 with 965/1029 amino acid identity (93%) to human FAK; synthetic FAK mutants, including the FAK Y397F (SEQ ID N0:2), K454R (SEQ ID N0:3), FRNK (an amino terminal truncant having FAK residues 694-1052 preceded by an initiator MET) (SEQ ID N0:4), and various phosphorylation mimics including FAK Y397D, Y397E, Y577D, Y577E, Y861 D, Y861 E, Y925D, Y925E and combinations thereof; CD2-FAK fusion (a constitutively active FAK fusion with CD2) described by in Chan P, et al., J Biol. Chem. (1994); 269 (32):
20567-74.
As used herein, the term "inducing agent" is an agent, compound, or chemical that produces a signal to noise ratio of at least 6-fold. Examples of inducing agents include but are not limited to Mifepristone (Ru486) and other antiprogestins such as Org31806 and Org31376. See O'Malley et. al., Cell, 69, 703-713 (1992). As used herein, the term capture agent is an agent, compound or chemical that is capable of capturing any form of focal adhesion kinase, including FAK tagged with histidine residues, streptavidin or other comparable affinity tags. The capture agent includes, but is not limited to, the phosphotyrosine FAKY'9' specific antibody, a general phosphotyrosine antibody, anti-FAK
antibody, anti-histidine tag antibody and molecules containing biotin which would facilitate the capture of streptavidin-modified FAK. In certain embodiments of the invention, the capture agent includes a combination of one or more antibodies, including but not limited to the combination of goat anti-rabbit antibody and the phosphotyrosine FAKY3s' specific antibody.
As used herein, the term "anti-phospho-tyrosine antibody" includes but is not limited to phosphotyrosine FAKY'9' specific antibody as well as general phosphotyrosine antibodies, where the latter are capable of recognizing any phosphorylated tyrosine residue, including but not limited to the tyrosine residue at position 397 of FAK.
As used herein, a cytoxicity indicator is an agent, chemical or compound that is used to assess cell viability. Examples of cytoxicity indicators include, but are not limited to tetrazolium salts (e.g. MTT, XTT, WST-1 ) that are especially useful for this type of analysis.
MTT is a yellow tetrazolium salt that is cleaved to purple formazan crystals by metabolic active cells. The solubilized formazan product may be spectrophotometrically quantified using an ELISA reader or other spectrophotometric device. The number of living directly correlates to the amount of purple formazan crystals formed, as monitored by the absorbance.
An embodiment of the present invention is carried out using a inducible FAK
gene expression system having the ability to provide consistent and tight regulation of gene expression. In an embodiment of the invention, an inducible FAK gene expression system is provided, which comprises a system for the regulated expression of a transgene, i.e., FAK.
FAK expression is "off' in the absence of an inducing agent, but "on" in its presence. The system consists of two genes: one which codes for a regulatory protein, and the other which codes for the inducible transgene of interest. Expression of the regulatory protein is driven by a constitutive promoter. The inducible FAK transgene has a promoter that consists of a minimal promoter linked to multiple copies of a binding site that is capable of binding the regulatory protein.
In an embodiment of the invention, the regulatory protein is a transcription factor that consists of the yeast GAL4 DNA binding domain, a truncated human progesterone receptor ligand binding domain, and the human p65 activation domain from NF-xB to facilitate tight regulation of the transgene over basal expression. The plasmid encoding the regulatory protein contains the GAL4 promoter which establishes a positive feedback loop to facilitate rapid response upon ligand treatment. The exogenous control of regulatory protein expression is achieved using a small molecule ligand. Tight regulation is achieved by species selective binding of the regulatory protein to the promoter of the transgene, and through ligand-dependent conformational activation of the regulatory protein.
Thus, in certain embodiments, the invention has the following advantages:
~ Induction of FAK in cells using an inducible system, which allows for tight repression of gene expression when not induced, resulting in viable cell clones.

~ Detection of phosphorylated FAK may be used to identify inhibitors of FAK
kinase activity.
Some of the methods of detection of phosphorylated FAK include Polyacrylamide gel electrophoresis-based assays and ELISA-based assays for identification of FAK
inhibitors. Other suitable detection systems may also be used.
~ The assay allows for detection of total FAK protein, total phosphorylated FAK protein, or FAK protein phosphorylated at a given tyrosine (e.g. tyrosine 397).
~ The cell-based system may be used for in vivo screening of FAK inhibitors, since the transfected cells are tumorigenic and FAK can be induced in vivo by feeding animals Mifepristone. The system's utility in vivo has been demonstrated.
COMPARATIVE EXAMPLES
Comparative Example 1 Attempts were made to generate stable ectopically expressed FAK or FAK mutant proteins in a variety of cell backgrounds in hopes of improving assay signal and noise. These efforts, however, also proved unsuccessful since most of these cells demonstrated sensitivity to changes in FAK protein levels and never formed viable clones with which to develop a cell-based assay. Cells expressing low to moderate levels of endogenous FAK such as, NIH3T3 mouse fibroblast or A2058 human metastatic melanoma cells tolerated no more than two-fold expression of FAK over endogenous levels. Consequently, these clones proved insufficient for development of the assay due to similar reasons described above: poor stimulation and reproducibility, high background noise, and not conducive to drug discovery.
Therefore, non-inducible expression of FAK in cells containing native FAK was not found to be suited for studying the induction of FAK expression.
Comparative Example 2 In order to develop a FAK cell-based assay that is both conducive to Medium and High Throughput Screening (ELISA system) and tracks the biochemical mechanism of FAK, a number of approaches were taken to exploit FAK biology. For example, attempts were made to mimic adherent cell stimulation of FAK by plating cells on Extracellular matrix proteins (ECM) proteins such as fibronectin, or collagen, or other ECMs such as matrigel, but this approach proved inefficient, resulted in poor stimulation of FAK, and was entirely dependent on cell type. Furthermore, ECM-matrigel-induced attachment of cells resulted in no stimulation of FAK phosphorylation as measured by pY54"RP (Horseradish Peroxidase (HRP)-conjugated phosphotyrosine antibody) detection, and control detection using secondary Anti-Mouse"RP (2°M"RP) alone resulted in nonspecific increases in assay signal in wells incubated with lysate derived from cells stimulated on matrigel. The signal and noise ratio (S/N) when using a combination of pY54"RP and 2°M"RP ranged from 1.0 to 1.7, while a combination of pY54 (unconjugated phosphotyrosine antibody) and 2°M"RP yielded a S/N
ratio of 1.0 to 2.4.
This suggested, and it was confirmed, that the nonspecific increase in assay signal was due -g_ to cross-reactivity to some matrigel contaminants) in the cell lysate. Hence, the above approach to develop an appropriate FAK cell-based assay was not viable.
Results also demonstrated a modest and suboptimal two- to three-fold signal over noise stimulation of FAK phosphorylation upon cell attachment to either ECM
matrigel or fibronectin. Further optimization of these assay conditions either by varying cell number, cell-ECM time for attachment, type of detection and capture antibodies, and combinatorial stimulation using Endothelin-1 and Phorbol 12-Myristate 13-Acetate (PMA) indicated that these non-inducible methods of FAK stimulation were insufficient, irreproducible, and not feasible for assay development.
Comparative Examble 3 Since integrin clustering leads to FAK phosphorylation, the feasibility of antibody crosslinking of integrin receptors using antibodies specific to ~i1 integrin was also examined.
However these efforts also proved to be unsuccessful because indirect methods of FAK
stimulation lead to high variability and irreproducibility. For example, integrin ~i1 cross-linking proved to be too cumbersome for even medium throughput screening since optimal stimulation required temperature sensitive steps that were time consuming and difficult to manage. Moreover, maintaining cells on polystyrene plates to lower background noise resulted in decrease cell viability due to poor ECM contact, thereby making these non-inducible expression systems infeasible for assay development.
Comparative Example 4 To advance the development of the FAK cell-based assay, an attempt was made to generate stable clones expressing either wild-type or mutant FAK proteins in a variety of cell backgrounds. Clones were generated that harbored FAK cDNA transcripts that encode either a constitutively active protein (CD2~FAK), a membrane bound tyrosine-dead protein (CD2~FAKY3s'F), or cytoplasmic FAK mutants that lack key downstream tyrosine residues (e.g., FAKY86'F, FAKY925F~ and FAKY867F/Y825F). These biological tools were strategically designed based on the knowledge of FAK biology in order to address the issues described above and to necessitate the development of a robust and reproducible cell-based assay conducive to drug discovery.
Attempts to generate stable clones overexpressing FAKWT were unsuccessful, even though viable control clones expressing either vector construct or the t_acZ
protein were obtained. Transfection of FAK mutant constructs designed either to shut down downstream phosphorylation of FAK while preserving FAK phosphorylation at Y397 and kinase activity (FAKY861F' FAKY925F~ and FAKYBS'~n~szsF), or to bypass the requirement for integrin receptors (CD2~FAKWT and CD2~FAKY39'F), also proved unsuccessful. In cells that expressed low to moderate levels of endogenous FAK, such as the human metastatic melanoma A2058, viable _g_ clones were identified. However, A2058~FAK~ clones exhibit only an approximate two-fold increase in FAK levels which proved insufficient for development of the FAK
cell based assay.
In addition to the work on exogenous stimulation, control over basal FAK
phosphorylation and attempts to ectopically express FAK clones, FAKwT and tyrosine-dead FAKYSS7F cDNAs were subjected to tetracycline control using the Tet-On/Tet-Off inducible systems. However, no FAKE or FAKY3s~F transfectants were detected. Since viable clones could not be identified, the level of "leakiness", i.e., inadequate regulation of basal expression, in the FAKWT or FAKY3s'F Tet-transfectants could not be determined. The Tet-OnT""/Tet-OffTM
systems demonstrated variable control over target protein expression, and therefore were not suited for the regulation of lethal genes like FAK.
WORKING EXAMPLES
The wild-type FAKWT (SEQ ID NO: 5), tyrosine-dead FAKY3s'F (SEQ ID NO: 6), kinase-dead FAK~45aR (SEQ ID NO: 7) and the dominate-negative FAK-related nonkinase (FRNK) (SEQ ID NO: 8) were cloned into the GeneSwitchTM pGeneVS/His A-vector backbone as either BamHl-Apal or Kpnl-Apal inserts. As used herein, the term "gene encoding FAK"
includes without limitation, SED ID NOS: 1-4. These constructs are based on the published sequence for human FAK at GenBank accession number L13616, see Whitney,G.S.
et. al., DNA Cell Biol. 12 (9), 823-830 (1993), and each construct was sequence confirmed and aligned against the published sequence. The DNA constructs were engineered to include specific 5' and 3' restriction sites that are unique and distinct over the prior art. In addition, the 3' ends of certain constructs were also engineered to include tagged epitopes such as V5 and histidine tags.
Several cell lines were selected for transfection including the NIH Swiss Mouse fibroblast NIH3T3 (ATCC Accession No. CCL-92), the human epidermoid carcinoma (ATCC Accession No. CRL1555), and the human glioblastoma astrocytoma U87MG
(ATCC
Accession No. HTB-14). Each cell line exhibits unique characteristics that makes its application favorable for overexpression of FAK. For example, NIH3T3 cells offer species specific overexpression of FAK and ease of transfectability, A431 cells express moderate levels of endogenous FAK and provide a tumor background more representative of a FAK
native environment, and U87MG cells lack the putative FAK negative regulator PTEN (a tumor suppressor phosphatase). Using Stratagene's GeneJammerT"" Transfection reagent, the pSwitch regulatory vector was co-transfected with either the pGeneve°torVSHis, or the pGeneFAKWTVSHis, or the pGeneFAK mutants (pGeneFAKKasaRVSHis, and pGeneFAKY3s~FV5His) into A431 cells. Similarly, NIH3T3 and U87MG cells were transformed to co-express the pSwitch regulatory protein and the specific pGene construct of interest.
Hygromycin and ~eocin resistant clones were selected and expanded in culture for screening via western and RT-PCR analysis. Several inducible clones including, A431:FAKWTVSHis, A431:FAKY3s'FVSHis, A431:FAKKasaRVSHis, A431:Vector, NIH3T3:FAKWTVSHis, NIH3T3:FAKY3s'FVSHis, N1H3T3:Vector, U87MG:FAKWTV5His, and U87MG:FAKY3s'FVSHis were successfully isolated, selected, confirmed and validated.
The FAK inducible assay of this embodiment of the invention measured FAK
phosphorylation at Y397. A431:FAKWTVSHis cells (about 1.0 x 10a to 1.0 x 10' cells) are seeded on U-bottom 96-well plates and allowed to attach at 37°C, 5% COZ
for 4 to 6 hoursprior to overnight incubation in the presence of 0.1 nM Mifepristone.
Subsequently, A431:FAKWTVSHis induced cells are either left untreated or treated with inhibitory compounds for 30 minutes at 37°C, 5% CO~ and lysed in RIPA lysis buffer (50 mM
Tris-HCI, pH7.4, 1%
NP-40, 0.25% Na-deoxycholate, 150 mM NaCI, 1 mM EDTA, 1 mM Na3VOa, 1 mM NaF, and one CompIeteT"" EDTA-free protease inhibitor pellet per 50 ml solution).
Approximately 45 Ng of total protein (100 NI) is then transferred to goat Anti-rabbit plates coated with 0.35 pglweil of Anti-FAK phosphospecific Y397 antibody for the capture and subsequent detection of phosphorylated FAK proteins (See Figure 3).
NIH3T3:FAKWTVSHis clones and NIH3T3:FAKY3s'FVSHis clones were seeded to approximately 80% confluency in T25 flasks and were either left untreated or stimulated with 0.1 nM Mifepristone for approximately 16 hoursat 37°G, 5%CO~. Cell lysates were prepared and immunodepleted of total FAK protein using the Anti-FAK CUBIT"', Lake Placid, N.Y.) monoclonal antibody. FAK immunocomplexes were then subject to SDS-Polyacrylamide gel electrophoresis and immunoblotted with Anti-FAK (A17) polyclonal antibody.
Table 1 shows densitometric quantitation measured in arbitrary "'band light" units of the autoradiographic film and fold changes in FAK protein levels as compared with unstimulated cells.
Table 1: Mifepristone induced FAK protein levels as measured by Densimetric Quantitation Clone FAKwt FAKwt FAKwt FAKwt number Mifepristone+ - + _ + _ + -Band Light2.OOE+061.OOE+068.OOE+05S.OOE+052.OOE+06S.OOE+053.OOE+06S.OOE+05 Units Fold 2 1 2 1 4 1 6 1 Stimulation It was observed that FAKWT inducible clones responded very well to mifepristone stimulation.
The above conditions translated easily to a 96-well ELISA format. Table 2 shows NIH3T3 clones induced to express FAKWT or FAKYSS7F proteins. FAKWT and FAKY3s'F proteins were captured either to measure total FAK protein or phosphotyrosine FAKY39' induced by mifepristone.
Table 2: Mifepristone induced FAK protein levels as measured by optical density 450 measurements NIH3T3FAK SignalNIH3T3FAKY397F Signal NIH3T3vector Signal (+) (+) (+) wt /Noise(-) /Noise(-) /Noise(-) Ratio Ratio Ratio Mifepristone.~.._ + - +

PhosphoFAK1.690.23 7 0.1 0.12 1 0.080.04 2 Y397 Capture FAK protein1.940.33 6 2.56 0.46 6 0.210.16 1 Capture It was observed that NIH3T3 clones yield a robust phosphotyrosine FAKY39' and FAK
protein signal-to-noise ratio of ~7 and -6, respectively. Both the NIH3T3 tyrosine-dead clone and the vector NIH3T3 transfectants confirm assay specificity for phosphoFAKY397.
Table 3 shows A431:FAK clones assayed under optimized conditions.
Table 3: Mifepristone induced FAK protein levels as measured by optical density 450 measurements A431:FAKwt Signal A31:FAK Signal A31:vector Signal (+) (+) (+) INoise(-)Y397F /Noise(-) /Noise(-) Ratio Ratio Ratio Mifeprist-I- - + - + -one Phospho2.09 0.1317 0.13 0.08 1.4 0.12 0.11 1.0 Capture FAK 2.22 0.2111 2.31 0.22 11 0.19 0.17 1.0 protein Capture Under optimal conditions, the A431:FAK clones yielded a robust phosphotyrosine FAKY39' and FAK protein signal-to-noise ratio of -17 and ~11, respectively.
Both the A431 tyrosine-dead clone and the vector A431 transfectants confirmed assay specificity for phosphoFAKY397. Induction of phosphotyrosine Y397 and FAK proteins were controllable by varying assay conditions such as mifepristone time of incubation, mifepristone concentration, cell density, and antibody concentrations.
The mechanism of FAK activation in A431 and other cell backgrounds was studied.
A431:FAKwT cells were either left untreated or stimulated with mifepristone.
First, a washout experiment was performed to demonstrate a time-dependent decrease in FAK
protein and phosphotyrosine Y397 levels (i.e. cells stimulated with mifepristone overnight (16hs) were subsequently cultured in fresh growth media in the absence of mifepristone over time).
Second, A431:FAK~ cells stimulated with mifepristone were detached and suspended in fresh growth media for 15 minutes followed by re-plating for 4, 24, 48, and 72 hourson tissue culture treated petri dishes. Table 4, experiment A, shows densimetric quantitation measured in arbitrary "band light" units of the autoradiographic film and fold changes in FAK
phosphorylation as compared to a no stimulation control.
Table 4: FAK phosphorylation as measured by Densimetric Quantitation Experiment A
A A431 FAKwt 4hrs 24 48 72 hrs hrs hrs Mifepristone + + + + +
-Band Light 5.7534 57.83277.37571.03327.719 92.834 Units Fold Increase1 10 13 12 5 16 over Uninduced Exueriment B
_~~

FAKwt 4hrs 24 48 72 hrs hrs hrs Mifepristone + + + + +
-Band Light 20723 44791 89444 6594611473 82273 Units Fold Increase1 2 4 3 1 4 over Uninduced In experiment B, a modest 4-fold induction over untreated cells was observed.
However, suspending stimulated cells for 15 minutes in fresh growth media followed by a 4-hour attachment to plastic led to a decrease in phosphoFAKY3sp. Moreover, allowing suspended mifepristone-induced cells to re-attach for 24 hours resulted in the re-activation of hos hot rosine FAKY3s' p p y , suggesting that the mechanism of FAK activation was intact.

Consistent with a time-dependent decrease in FAK protein levels, phosphotyrosine FAKY39' decreased to unstimulated control levels by 72 hours.
Cellular suspension leads to inactivation of FAK phosphorylation. Re-activation of FAK may be achieved by re-plating suspended cells to allow cell attachment.
Table 4 demonstrates that the mechanism of FAK activation is intact under the regulatory system discussed above. In other words, exogenous stimulation with mifepristone leads to the activation of FAK that is mechanistically relevant.
The FAK inducible cell-based assay of the invention was successfully employed in the identification of a number of FAK inhibitors, including PP1 and staurosporin. In addition, the assay was used to determine the cytoxicity of particular compounds on FAK-expressing cells. In these experiments, the test compound was placed in contact with FAK-induced or uninduced control cells. Table 5 shows changes in FAK phosphorylation in OD4so units upon treatment with increasing concentration of a FAK inhibitor. Table 6 shows densimetric quantitation measured in arbitrary "band light" units of the autoradiographic film and fold changes in FAK phosphorylation in the presence of increasing concentration of the FAK
inhibitor. Thus, the assay of the invention can be advantageously used in a FAK drug discovery program.
The FAK inducible cell-based assay was used as described in Figure 3 to screen a FAK inhibitor. A 10 NM curve of an FAK inhibitor was performed in 1/2 log dilutions as shown in Table 5. The % inhibition was determined and the inhibition concentration at 50% (ICSO) was calculated. A431:FAK~ cells were seeded in T25 flask to ~80% confluency and were either left untreated or stimulated overnight with 0.1 nM mifepristone.
Subsequently, stimulated cells were treated with the same FAK inhibitor as in Table 5 at the same 1/2 log concentrations of a 10 pM curve for 30 minutes at 37°C, 5% COz. Cell lysates were then prepared and total protein concentration determined by protein assay.
Equivalent amounts of total protein were subject to SDS-Polyacrylamide gel electrophoresis and western analysis using the phosphotyrosine FAKY39' specific antibody, the general phosphotyrosine pY20 antibody, and the Anti-FAK (A17) antibody.
Table 5: FAK phosphorylation as measured by optical density 450 measurements Noise FAK
Inhibitor+A431 FAKwt WT 10 NM 3.3 1.1 0.37 0.12 0 pM
pM NM pM NM

Mifepristone- + + + + + +

PhosphoFAKY390.28 0.56 0.98 1.65 2.21 2.56 2.77 7 (OD450) Minus 0.000 0.28 0.70 1.37 1.93 2.28 2.49 Noise FAK
Inhibitor+A431FAKwt WT 10 3.3 1.1 0.37 0.12 0 ~M
NM pM ~M NM pM

Background %Control 11 28 55 78 92 100 %Inhibition 89% 72% 45% 22% 8% 0%

The calculated Inhibition Concentration at 50% for the FAK inhibitor was -0.93 uM.
Table 6 shows densimetric quantitation measured in arbitrary "band light"
units of the autoradiographic film and fold changes in FAK phosphorylation in the presence of increasing concentration of the FAK inhibitor. These experiments further demonstrate the usefulness of the assay of the invention in a FAK drug discovery program.
Table 6: FAK phosphorylation as measured by Densimetric. Quantitation A: IB:AntiFAK(A17) BackgroundFAK
Inhibitor+A431 FAKwt WT 10 3.3 pM 1.1 pM 0.37 0.12 0 pM NuM pM pM

Mifepristone_ + + + + + +

BandLight30.501 119.6 100.2 108.3 128.1 90.087 82.414 Units Minus 0 89 70 78 98 60 52 Background %Control 172 134 150 188 115 100 %Inhibition 0% 0% 0% 0% 0% 0%

B: IB:AntiFAKpY397 BackgroundFAK
Inhibitor+A431 FAKwt WT 10 3.3 1.1 0.37 NM 0.12 0 NM
NM NM pM NM

Mifepristone- + + + + + +

Band Light0.34513 3.923810.44 20.21 45.27 64.56 68.022 Units Minus 0 4 10 20 45 64 68 Background %Control 5 15 29 66 95 100 %Inhibition 95% 85% 71% 34% 5% 0%

C: IB:AntipY20 BackgroundFAK
Inhibitor+A431 FAKwt WT 10 3.3 1.1 pM 0.37 0.12 0 pM NM pM NM NM

Mifepristone_ + + + + + +

Band Light0.7577 5.22069.058 21.51 25.19 26.323 33.1 Units 32 Minus 0 4 8 21 24 26 32 Background %Control 14 26 64 75 79 100 %InhibitionI 86% 74% 36% ~ 25% 21 % 0 I ' I

As a control for protein loading, the amounts of FAK protein in experiment A
were equivalent and incubation with the FAK inhibitor did not have any effect on FAK expression.
In experiment B, consistent with the IC50 reported in Table 5, an Anti-FAKpY397 blot showed an estimated IC50 value in the range of 0.37 and 1.1 uM, where the 50%
concentration is closer to 1.1 NM. As seen in experiment C, similarly and consistent with FAK
biology, total FAK phosphorylation as measured by an Anti-pY20 immunoblot was inhibited by the inhibitor in an estimate IC50 range of 1.1 to 3.3 NM, where the 50% inhibition concentration was closer to 3.3 pM.
Exogenous Control of FAK ohospho Ir a ECM stimulation of FAK was achieved via plating 2.0x105 cells/well (e.g.
A2058) on commercially available fibronectin (FN) 96-well plates or on FN 96-well plates prepared in-house. Cells were allowed to attach for 15, 30, 60, or 90 minutes at 37°C, 5%COZ to induce integrin engagement and subsequent FAK phosphorylation. Cells lysates were prepare in RIPA lysis buffer (50 mM Tris-HCI, pH7.4, 1 % NP-40, 0.25% Na-deoxycholate, 150 mM NaCI, 1 mM EDTA, 1 mM Na3V04, 1 mM NaF, and one CompIeteTM EDTA-free protease inhibitor pellet per 50 ml solution) and transferred to Anti-FAK coated 96-well plates to capture total FAK protein. Phosphotyrosine FAK proteins were measured using the general phosphotyrosine antibody Py54. Similarly, cells were stimulated on commercially available matrigel coated plates or on matrigel plates prepared in-house. For western analysis, cells were allowed to attach to either commercially available ECM coated flask or to ECM coafied flask prepared in-house for the indicated times above. Cell lysates were prepared in RIPA
iysis buffer and total and phosphotyrosine FAK proteins were either pull-down or immunodetected using Anti-FAK antibodies or Anti-phosphotyrosine antibodies.
Cells (e.g. A2058 human metastatic meloanomas) were seeded in growth media DMEM10%FBS, Pen/Strep/Glu ) on 96-well plates or T25/T75 flasks and allowed to adhere to tissue culture treated plastic for approximately five hours at 37°C, 5% CO~. Subsequently, they were starved in 0.1 % FBS DMEM starvation media overnight at 37°C, 5% C02, followed by stimulation via treatment with (up to 100 NM) Endothelin I, (up to 100 nM) Bombesin, or (up to 800 nM) PMA. Exposure to cytokines varied from 10 minutes to as long as 60 minutes at 37°C, 5% CO2. Total FAK proteins were either captured in ELISA format or immunoprecipitated using Anti-FAK specific antibodies. Phosphotyrosine FAK
proteins were then detected with a general Anti-phopshotyrosine antibody either in ELISA or Western format.
Starved Cells (e.g. A2058) were pre-chilled at 4°C for 30 minutes prior to incubation with varying concentrations (1:100, 1:330, 1:1000, 1:3,300, or 1:10,000 of stock concentration of 200 Ng/ml) of a1-Integrin antibody (4° C, 30 minutes). (31-Integrin clustering was induced with 1:500 dilution of Goat anti-mouse secondary antibody for 30 minutes at 4°C. Whole cell lysates were then prepared in lysis buffer (10 mM Tris-HCI, 5-mM NaCI, 10 mM
EDTA, 2mM
Na Vanadate, 1%NP40, protease inhibitors), and total FAK proteins were immunoprecipitated using Anti-FAK antibody. Phosphotyrosine FAK proteins were measured using the Anti-phosphotyrosine antibody Py54.
Clonin_a of FAKWT and FAK mutants (Noninducible and Tet-OnTM/Tet-OffTM
Systems) The full-length FAKWT cDNA was cloned from a T-cell cDNA library using the primers FAKS'Bam: GGATCCATGGCAGCTGCTTACCTTGAC (SEQ ID NO.: 9) and FAK3'Bam:
GGATCCTCACTCACTCAGTGTGGTCTCGTCTGCCCA (SEO ID NO.: 10) in RT-PCR and was sequence confirmed against the accession number L13616. Site-directed mutagenesis of the FAKWT template was performed using the Stratagene QuikChangeT"" Site-Directed Mutagenesis Kit. To generate plasmids for electroporation into Clontech pre-made HeLa or HEK293 tet-On/tet-Off cell lines, the full-length FAKWT, the tyrosine-dead mutant (FAKY3a~F), and the kinase-dead mutant (FAK~454R) cDNAs were subcloned into the BamHl sites of the pTRE:FLAG vector. The following constructs were generated and electroporated into HeLa Tet-Off, HeLa Tet-On, or 293 Tet-On: pTRE:FLAG Vector, pTRE:FAK~"'~FLAG, and pTRE:FAKY3s~FFLAG. 6418 resistant clones were selected and expanded in DMEM+100ug/ml 6418 growth media.
Clonin4 of FAKWT and FAK mutants (GeneSwitchTM System) The full-length Focal Adhesion Kinase (FAKWT) cDNA was cloned from a T-cell cDNA
library using the above primers in RT-PCR and was sequence confirmed against the accession number L13616. Site-directed mutagensis of the FAK~"'~ template was performed using the Stratagene QuikChangeT"" Site-Directed Mutagenesis Kit. The full-length FAKWT, the tyrosine-dead mutant (FAKY39'F), and the kinase-dead mutant (FAKK~54~) cDNAs were subcloned into the BamHl-Apal sites of the pGeneVS/His-A plasmid. The dominate-negative FAK-related nonkinase (FRNK) cDNA was subcloned as a Kpnl-Apal insert into the cassette of the pGeneVS/His A-vector. These plasmids were confirmed via DNA sequencing and restriction digestion analysis. Subsequently, these constructs were co-transfected with the pSwitch construct encoding the GeneSwitchT"" Protein into NIH3T3, A431, or U87MG cells using Stratagene's GeneJammerT"" Transfection reagent. Transfectants were grown and selected in DMEM growth media (10%FBS, Pen/Strep/Glu) spiked with 750 pg/ml of Zeocin and 50 Ng/ml of hygromycin antibiotics. Hygromycin and Zeocin resistant clones were selected and expanded in culture for screening in both Western and RT-PCR
format.
Screening of A431 ~FAK clones A431~FAKWT and A431~FAKY3s'F clones were seeded in T25 flasks to near 80%
confluency and were either left untreated or treated with 0.1 nM of Mifepristone ligand overnight (-16hrs). A431 transfectants were then lysed in RIPA lysis buffer (50 mM Tris-HCI, pH7.4, 1 % NP-40, 0.25% Na-deoxycholate, 150 mM NaCI, 1 mM EDTA, 1 mM Na3V04, 1 mM
NaF, and one CompIeteTM EDTA-free protease inhibitor pellet per 50 ml solution) and cell lysates were assayed for total protein concentration. Equivalent total protein concentrations were subject to western analysis immunoblotting for FAK, or FAK mutant, proteins and phosphotyrosine FAK'~39' using standard western blotting techniques. Clones tested positive and exhibit at least 3X induction over endogenous FAK levels were selected for optimization and development of the FAK cell-based assay.
Clones were also screened and verified via RT-PCR. Cytoplasmic mRNA
transcripts were isolated and purified from A431 ~FAK wild-type and mutant clones for cDNA
synthesis using random hexamers. Polymerase chain reactions were subsequently performed on cDNA libraries derived from A431 ~FAK transfectants using primers specific to the N-terminus, the internal FAK sequences, and the C-terminus of FAK. Primers specific to the antibiotic transcripts Zeocin or Hygromycin and GAPDH were also used to amplify these genes as internal controls for transfection as well as for the quality of cDNA
libraries.
Optimization of FAK inducible cell-based System A431 ~pGeneve't°r, A431 ~FAKWT, A431 ~FAKx454~, and A431 ~FAKY3s'F
clones were seeded in T25 flasks to near 80% confluency and were either left untreated or treated with 0.1, 10, or 100 nM of Mifepristone ligand overnight (~16hrs). A431 transfectants were then lysed in RIPA lysis buffer (described above) and cell lysates were assayed for total protein concentration. Equivalent total protein concentrations were subject to western analysis immunoblotting for FAK, or FAK mutant, proteins and phosphotyrosine FAKYas7 using standard western blotting techniques. To determine that optimization of Mifepristone concentration in western format translates into an ELISA system, Mifepristone concentration and time of incubation were also optimized in ELISA format.
A431 ~pGene~e°t°', A431 ~FAK~"'~, and A431 ~FAK"3s'F clones were seeded in 96-well U-bottom plates at a cell density of 1.2 x 106 cells/ml. Cells were allowed to sit at 37°C, 5%

COZ for 6 to 8 hours prior to FAK induction with Mifepristone ligand. A431 clones were subsequently left untreated or treated with 0.1, 10, 100, or 110 nM of Mifepristone for ~0.5, 1.0, 2.0, 4.0 or 24.0 hoursat 37°C, 5% CO~. Cells were then lysed in RIPA lysis buffer and 100 pl of cell lysate (45 Ng total protein) were transferred to 96 well plates. FAK or FAK
mutant proteins and phosphotyrosine FAKY3s~ were subsequently captured on goat anti-mouse or goat anti-rabbit plates coated with either FAK specific antibodies or phosphotyrosine FAKY3s' antibodies, respectively. To capture FAK or FAK mutant proteins, for example, goat anti-mouse plates were coated with 0.5pg/ml of Anti-FAK
(UBI) monoclonal antibodies prior to incubation with 100 pl of cell lysafie (45 pg total protein). Detection of captured FAK or FAK mutant proteins were then measured by Anti-FAK (A17) polyclonal antibody. Similarly, capture of phosphotyrosine FAKv3sr proteins were performed via coating goat anti-rabbit plates with 3.5 Ng/ml of Anti-FAKp[Y397] polyclonal antibody, followed by detection using Anti-FAK (UBI) monoclonal antibody.
Parameters can be optimized for the particular high thoughput screening system employed, including evaluating the types of 96-well plate (e.g., anti-rabbit, protein A, or protein G), capture antibody concentration, detection antibody concentration, secondary horseradish peroxidase (HRP) antibody concentration, cell density, blocking buffer (e.g.
SuperBlock Blocking TBS, 3%BSA blocking), and time of compound treatment.
Using purified GST~FAK protein that is phosphorylated at residue Y397, optimization of capture antibody concentration was first determined by coating 96-well Anti-rabbit plates with increasing concentrations of anti-FAKp[Y397] phosphospecific antibody. A
plot of captured phosphospecific GST~FAKY38' vs. Anti-FAKp[Y397] antibody concentration was generated to determine the optimal capture antibody concentration. Using this preliminary Anti-FAKp[Y397] capture concentration, the other parameters (detection and secondary antibody concentrations, cell density, blocking buffer) were optimized and formatted for High Throughput Screening (HTS). Most of these parameters were optimized or evaluated simultaneously either by establishing a 96-well matrix or by cross-comparing changes in signal-to-noise ratios among a number of permutations. For example, on 96-well Anti-rabbit plates coated with 3.5 Ng/ml of Anti-FAKp[Y397] capture antibody, increasing concentrations of Detection and Secondary"RP antibodies were set-up in a 96-well matrix to detect captured phosphotyrosine FAKYas~ proteins to optimized the concentrations of these antibodies. This experiment may be repeated on Protein A plates and/or by varying blocking buffer or other parameters to cross compare the signal-to-noise values among the number of permutations to optimize the system.
Validation of FAK cell based assay A431~FAKWT cells were seeded in T25 flasks to near 80% confluency and were either left untreated or treated with 0.1 nM of Mifepristone ligand overnight (~16hrs). A431 ~FAK""~

uninduced and induced cells were washed with 10 ml of PBS and suspended in 15 ml growth media (DMEM 10%FBS, Pen/Strep/Glu, 750 Ng/ml Zeocin, 50 ug/ml Hygromycin) for 30, 60, 90, or 120 minutes at 37°C, 5% CO2. Cells were subsequently lysed in RIPA lysis buffer (described above) and cell lysates were assayed for total protein concentration. Equivalent total protein concentrations were subject to western analysis immunoblotting for FAK or phosphotyrosine FAKY3s' proteins using standard western blotting techniques.
A431 ~FAK"~'~ and A431 ~FAKY3s'F clones were seeded in T25 flasks to near 80%
confluency and were either left untreated or treated with 0.1 nM of Mifepristone ligand overnight (~16hrs). A431~FAKWT cells were subsequently treated with increasing concentrations of a FAK inhibitor (half-log dilutions of a 10 NM-starting solution) identified by using the FAK inducible cell-based assay. Cell lysates were then prepared in RIPA lysis buffer and assayed for total protein concentration. Equivalent total protein concentrations were subject to western analysis immunoblotting for FAK, general phosphotyrosine FAK, or phosphotyrosine FAKY39' proteins using standard western blotting techniques.

SEQUENCE LISTING
PC11699A.ST25.txt <110> Roberts, Walter Gregory Ung, Ethan James Tekly Whalen, Pamela Mathews <120> INDUCIBLE FOCAL ADHESION KINASE CELL ASSAY
<130> PC11699A
<160> 10 <170> Patentln version 3.2 <210> 1 <211> 1052 <212> PRT
<213> Human wild type FAK (V5 epitope and His tag not included) <400> 1 Met Ala Ala Ala Tyr Leu Asp Pro Asn Leu Asn His Thr Pro Asn Ser Ser Thr Lys Thr His Leu Gly Thr Gly Met Glu Arg Ser Pro Gly Ala Met Glu Arg Val Leu Lys Val Phe His Tyr Phe Glu Ser Asn Ser Glu Pro Thr Thr Trp Ala Ser Ile Ile Arg His Gly Asp Ala Thr Asp Val Arg Gly Ile Ile Gln Lys Ile Val Asp Ser His Lys Val Lys His Val Ala Cys Tyr Gly Phe Arg Leu Ser His Leu Arg Ser Glu Glu Val His Trp Leu His Val Asp Met Gly Val Ser Ser Val Arg Glu Lys Tyr Glu Leu Ala His Pro Pro Glu Glu Trp Lys Tyr Glu Leu Arg Ile Arg Tyr Leu Pro Lys Gly Phe Leu Asn Gln Phe Thr Glu Asp Lys Pro Thr Leu Asn Phe Phe Tyr Gln Gln Val Lys Ser Asp Tyr Met Leu Glu Ile Ala PC11699A.ST25.txt Asp Gln Val Asp Gln Glu Ile Ala Leu Lys Leu Gly Cys Leu Glu Ile Arg Arg Ser Tyr Trp Glu Met Arg Gly Asn Ala Leu Glu Lys Lys Ser Asn Tyr Glu Val Leu Glu Lys Asp Val Gly Leu Lys Arg Phe Phe Pro Lys Ser Leu Leu Asp Ser Val Lys Ala Lys Thr Leu Arg Lys Leu Ile Gln Gln Thr Phe Arg Gln Phe Ala Asn Leu Asn Arg Glu Glu Ser Ile Leu Lys Phe Phe Glu Ile Leu Ser Pro Val Tyr Arg Phe Asp Lys Glu Cys Phe Lys Cys Ala Leu Gly Ser Ser Trp Ile Ile Ser Val Glu Leu Ala Ile Gly Pro Glu Glu Gly Ile Ser Tyr Leu Thr Asp Lys Gly Cys Asn Pro Thr His Leu Ala Asp Phe Thr Gln Val Gln Thr Ile Gln Tyr Ser Asn Ser Glu Asp Lys Asp Arg Lys Gly Met Leu Gln Leu Lys Ile Ala Gly Ala Pro Glu Pro Leu Thr Val Thr Ala Pro Ser Leu Thr Ile Ala Glu Asn Met Ala Asp Leu Ile Asp Gly Tyr Cys Arg Leu Val Asn Gly Thr Ser Gln Ser Phe Ile Ile Arg Pro Gln Lys Glu Gly Glu Arg Ala Leu Pro Ser Ile Pro Lys Leu Ala Asn Ser Glu Lys Gln Gly Met Arg Thr His Ala Val Ser Val Ser Glu Thr Asp Asp Tyr Ala Glu Ile PC11699A.ST25.txt Ile Asp Glu Glu Asp Thr Tyr Thr Met Pro Ser Thr Arg Asp Tyr Glu Ile Gln Arg Glu Arg Ile Glu Leu Gly Arg Cys Ile Gly Glu Gly Gln Phe Gly Asp Val His Gln Gly Ile Tyr Met Ser Pro Glu Asn Pro Ala Leu Ala Val Ala Ile Lys Thr Cys Lys Asn Cys Thr Ser Asp Ser Val Arg Glu Lys Phe Leu Gln Glu Ala Leu Thr Met Arg Gln Phe Asp His Pro His Ile Val Lys Leu Ile Gly Val Ile Thr Glu Asn Pro Val Trp Ile Ile Met Glu Leu Cys Thr Leu Gly Glu Leu Arg Ser Phe Leu Gln Val Arg Lys Tyr Ser Leu Asp Leu Ala Ser Leu Ile Leu Tyr Ala Tyr Gln Leu Ser Thr Ala Leu Ala Tyr Leu Glu Ser Lys Arg Phe Val His Arg Asp Ile Ala Ala Arg Asn Val Leu Val Ser Ser Asn Asp Cys Val Lys Leu Gly Asp Phe Gly Leu Ser Arg Tyr Met Glu Asp Ser Thr Tyr Tyr Lys Ala Ser Lys Gly Lys Leu Pro Ile Lys Trp Met Ala Pro Glu Ser Ile Asn Phe Arg Arg Phe Thr Ser Ala Ser Asp Val Trp Met Phe Gly Val Cys Met Trp Glu Ile Leu Met His Gly Val Lys Pro Phe Gln PC11699A.ST25.txt Gly Val Lys Asn Asn Asp Val Ile Gly Arg Ile Glu Asn Gly Glu Arg Leu Pro Met Pro Pro Asn Cys Pro Pro Thr Leu Tyr Ser Leu Met Thr Lys Cys Trp Ala Tyr Asp Pro Ser Arg Arg Pro Arg Phe Thr Glu Leu Lys Ala Gln Leu Ser Thr Ile Leu Glu Glu Glu Lys Ala Gln Gln Glu Glu Arg Met Arg Met Glu Ser Arg Arg Gln Ala Thr Val Ser Trp Asp Ser Gly Gly Ser Asp Glu Ala Pro Pro Lys Pro Ser Arg Pro Gly Tyr Pro Ser Pro Arg Ser Ser Glu Gly Phe Tyr Pro Ser Pro Gln His Met Val Gln Thr Asn His Tyr Gln Val Ser Gly Tyr Pro Gly Ser His Gly Ile Thr Ala Met Ala Gly Ser Ile Tyr Pro Gly Gln Ala Ser Leu Leu Asp Gln Thr Asp Ser Trp Asn His Arg Pro Gln Glu Ile Ala Met Trp Gln Pro Asn Val Glu Asp Ser Thr Val Leu Asp Leu Arg Gly Ile Gly Gln Val Leu Pro Thr His Leu Met Glu Glu Arg Leu Ile Arg Gln Gln Gln Glu Met Glu Glu Asp Gln Arg Trp Leu Glu Lys Glu Glu Arg Phe Leu Lys Pro Asp Val Arg Leu Ser Arg Gly Ser Ile Asp Arg Glu Asp Gly Ser Leu Gln Gly Pro Ile Gly Asn Gln His Ile Tyr Gln Pro Val PC11699A.ST25.txt Gly Lys Pro Asp Pro Ala Ala Pro Pro Lys Lys Pro Pro Arg Pro Gly Ala Pro Gly His Leu Gly Ser Leu Ala Ser Leu Ser Ser Pro Ala Asp Ser Tyr Asn Glu Gly Val Lys Leu Gln Pro Gln Glu Ile Ser Pro Pro Pro Thr Ala Asn Leu Asp Arg Ser Asn Asp Lys Val Tyr Glu Asn Val Thr Gly Leu Val Lys Ala Val Ile Glu Met Ser Ser Lys Ile Gln Pro Ala Pro Pro Glu Glu Tyr Val Pro Met Val Lys Glu Val Gly Leu Ala Leu Arg Thr Leu Leu Ala Thr Val Asp Glu Thr Ile Pro Leu Leu Pro Ala Ser Thr His Arg Glu Ile Glu Met Ala Gln Lys Leu Leu Asn Ser Asp Leu Gly Glu Leu Ile Asn Lys Met Lys Leu Ala Gln Gln Tyr Val Met Thr Ser Leu Gln Gln Glu Tyr Lys Lys Gln Met Leu Thr Ala Ala His Ala Leu Ala Val Asp Ala Lys Asn Leu Leu Asp Val Ile Asp Gln Ala Arg Leu Lys Met Leu Gly Gln Thr Arg Pro His <210> 2 <211> 1052 <212> PRT
<213> Human FAK Y397F mutant (V5 epitope and His tag not included) <400> 2 Met Ala Ala Ala Tyr Leu Asp Pro Asn Leu Asn His Thr Pro Asn Ser PC11699A.ST25.txt Ser Thr Lys Thr His Leu Gly Thr Gly Met Glu Arg Ser Pro Gly Ala Met Glu Arg Val Leu Lys Va) Phe His Tyr Phe Glu Ser Asn Ser Glu Pro Thr Thr Trp Ala Ser Ile Ile Arg His Gly Asp Ala Thr Asp Val Arg Gly Ile Ile Gln Lys Ile Val Asp Ser His Lys Val Lys His Val Ala Cys Tyr Gly Phe Arg Leu Ser His Leu Arg Ser Glu Glu Val His Trp Leu His Val Asp Met Gly Val Ser Ser Val Arg Glu Lys Tyr Glu Leu Ala His Pro Pro Glu Glu Trp Lys Tyr Glu Leu Arg Ile Arg Tyr Leu Pro Lys Gly Phe Leu Asn Gln Phe Thr Glu Asp Lys Pro Thr Leu Asn Phe Phe Tyr Gln Gln Val Lys Ser Asp Tyr Met Leu Glu Ile Ala Asp Gln Val Asp Gln Glu Ile Ala Leu Lys Leu Gly Cys Leu Glu Ile Arg Arg Ser Tyr Trp Glu Met Arg Gly Asn Ala Leu Glu Lys Lys Ser Asn Tyr Glu Val Leu Glu Lys Asp Val Gly Leu Lys Arg Phe Phe Pro Lys Ser Leu Leu Asp Ser Val Lys Ala Lys Thr Leu Arg Lys Leu Ile Gln Gln Thr Phe Arg Gln Phe Ala Asn Leu Asn Arg Glu Glu Ser Ile Leu Lys Phe Phe Glu Ile Leu Ser Pro Val Tyr Arg Phe Asp Lys Glu PC 11699A. ST25. txt Cys Phe Lys Cys Ala Leu Gly Ser Ser Trp Ile Ile Ser Val Glu Leu Ala Ile Gly Pro Glu Glu Gly Ile Ser Tyr Leu Thr Asp Lys Gly Cys Asn Pro Thr His Leu Ala Asp Phe Thr Gln Val Gln Thr Ile Gln Tyr Ser Asn Ser Glu Asp Lys Asp Arg Lys Gly Met Leu Gln Leu Lys Ile Ala Gly Ala Pro Glu Pro Leu Thr Val Thr Ala Pro Ser Leu Thr Ile Ala Glu Asn Met Ala Asp Leu Ile Asp Gly Tyr Cys Arg Leu Val Asn Gly Thr Ser Gln Ser Phe Ile Ile Arg Pro Gln Lys Glu Gly Glu Arg Ala Leu Pro Ser Ile Pro Lys Leu Ala Asn Ser Glu Lys Gln Gly Met Arg Thr His Ala Val Ser Val Ser Glu Thr Asp Asp Phe Ala Glu Ile Ile Asp Glu Glu Asp Thr Tyr Thr Met Pro Ser Thr Arg Asp Tyr Glu Ile Gln Arg Glu Arg Ile Glu Leu Gly Arg Cys Ile Gly Glu Gly Gln Phe Gly Asp Val His Gln Gly Ile Tyr Met Ser Pro Glu Asn Pro Ala Leu Ala Val Ala Ile Lys Thr Cys Lys Asn Cys Thr Ser Asp Ser Val Arg Glu Lys Phe Leu Gln Glu Ala Leu Thr Met Arg Gln Phe Asp His Pro His Ile Val Lys Leu Ile Gly Val Ile Thr Glu Asn Pro Val Trp PC11699A.ST25.txt Ile Ile Met Glu Leu Cys Thr Leu Gly Glu Leu Arg Ser Phe Leu Gln Val Arg Lys Tyr Ser Leu Asp Leu Ala Ser Leu Ile Leu Tyr Ala Tyr Gln Leu Ser Thr Ala Leu Ala Tyr Leu Glu Ser Lys Arg Phe Val His Arg Asp Ile Ala Ala Arg Asn Val Leu Val Ser Ser Asn Asp Cys Val Lys Leu Gly Asp Phe Gly Leu Ser Arg Tyr Met Glu Asp Ser Thr Tyr Tyr Lys Ala Ser Lys Gly Lys Leu Pro Ile Lys Trp Met Ala Pro Glu Ser Ile Asn Phe Arg Arg Phe Thr Ser Ala Ser Asp Val Trp Met Phe Gly Val Cys Met Trp Glu Ile Leu Met His Gly Val Lys Pro Phe Gln Gly Val Lys Asn Asn Asp Val Ile Gly Arg Ile Glu Asn Gly Glu Arg Leu Pro Met Pro Pro Asn Cys Pro Pro Thr Leu Tyr Ser Leu Met Thr Lys Cys Trp Ala Tyr Asp Pro Ser Arg Arg Pro Arg Phe Thr Glu Leu Lys Ala Gln Leu Ser Thr Ile Leu Glu Glu Glu Lys Ala Gln Gln Glu Glu Arg Met Arg Met Glu Ser Arg Arg Gln Ala Thr Val Ser Trp Asp Ser Gly Gly Ser Asp Glu Ala Pro Pro Lys Pro Ser Arg Pro Gly Tyr PC11699A.ST25.txt Pro Ser Pro Arg Ser Ser Glu Gly Phe Tyr Pro Ser Pro Gln His Met Val Gln Thr Asn His Tyr Gln Val Ser Gly Tyr Pro Gly Ser His Gly Ile Thr Ala Met Ala Gly Ser Ile Tyr Pro Gly Gln Ala Ser Leu Leu Asp Gln Thr Asp Ser Trp Asn His Arg Pro Gln Glu Ile Ala Met Trp Gln Pro Asn Val Glu Asp Ser Thr Val Leu Asp Leu Arg Gly Ile Gly Gln Val Leu Pro Thr His Leu Met Glu Glu Arg Leu Ile Arg Gln Gln Gln Glu Met Glu Glu Asp Gln Arg Trp Leu Glu Lys Glu Glu Arg Phe Leu Lys Pro Asp Val Arg Leu Ser Arg Gly Ser Ile Asp Arg Glu Asp Gly Ser Leu Gln Gly Pro Ile Gly Asn Gln His Ile Tyr Gln Pro Val Gly Lys Pro Asp Pro Ala Ala Pro Pro Lys Lys Pro Pro Arg Pro Gly Ala Pro Gly His Leu Gly Ser Leu Ala Ser Leu Ser Ser Pro Ala Asp Ser Tyr Asn Glu Gly Val Lys Leu Gln Pro Gln Glu Ile Ser Pro Pro Pro Thr Ala Asn Leu Asp Arg Ser Asn Asp Lys Val Tyr Glu Asn Val Thr Gly Leu Val Lys Ala Val Ile Glu Met Ser Ser Lys Ile Gln Pro Ala Pro Pro Glu Glu Tyr Val Pro Met Val Lys Glu Val Gly Leu Ala PC11699A.ST25.txt Leu Arg Thr Leu Leu Ala Thr Val Asp Glu Thr Ile Pro Leu Leu Pro Ala Ser Thr His Arg Glu Ile Glu Met Ala Gln Lys Leu Leu Asn Ser Asp Leu Gly Glu Leu Ile Asn Lys Met Lys Leu Ala Gln Gln Tyr Val Met Thr Ser Leu Gln Gln Glu Tyr Lys Lys Gln Met Leu Thr Ala Ala His Ala Leu Ala Val Asp Ala Lys Asn Leu Leu Asp Val Ile Asp Gln Ala Arg Leu Lys Met Leu Gly Gln Thr Arg Pro His <210> 3 <211> 1052 <212> PRT
<213> Human FAIC K454R mutant (V5 epitope and His tag not included) <400> 3 Met Ala Ala Ala Tyr Leu Asp Pro Asn Leu Asn His Thr Pro Asn Ser Ser Thr Lys Thr His Leu Gly Thr Gly Met Glu Arg Ser Pro Gly Ala Met Glu Arg Val Leu Lys Val Phe His Tyr Phe Glu Ser Asn Ser Glu Pro Thr Thr Trp Ala Ser Ile Ile Arg His Gly Asp Ala Thr Asp Val Arg Gly Ile Ile Gln Lys Ile Val Asp Ser His Lys Val Lys His Val Ala Cys Tyr Gly Phe Arg Leu Ser His Leu Arg Ser Glu Glu Val His Trp Leu His Val Asp Met Gly Val Ser Ser Val Arg Glu Lys Tyr Glu PC11699A.ST25.txt Leu Ala His Pro Pro Glu Glu Trp Lys Tyr Glu Leu Arg Ile Arg Tyr Leu Pro Lys Gly Phe Leu Asn Gln Phe Thr Glu Asp Lys Pro Thr Leu Asn Phe Phe Tyr Gln Gln Val Lys Ser Asp Tyr Met Leu Glu Ile Ala Asp Gln Val Asp Gln Glu Ile Ala Leu Lys Leu Gly Cys Leu Glu Ile Arg Arg Ser Tyr Trp Glu Met Arg Gly Asn Ala Leu Glu Lys Lys Ser Asn Tyr Glu Val Leu Glu Lys Asp Val Gly Leu Lys Arg Phe Phe Pro Lys Ser Leu Leu Asp Ser Val Lys Ala Lys Thr Leu Arg Lys Leu Ile Gln Gln Thr Phe Arg Gln Phe Ala Asn Leu Asn Arg Glu Glu Ser Ile Leu Lys Phe Phe Glu Ile Leu Ser Pro Val Tyr Arg Phe Asp Lys Glu Cys Phe Lys Cys Ala Leu Gly Ser Ser Trp Ile Ile Ser Val Glu Leu Ala Ile Gly Pro Glu Glu Gly Ile Ser Tyr Leu Thr Asp Lys Gly Cys Asn Pro Thr His Leu Ala Asp Phe Thr Gln Val Gln Thr Ile Gln Tyr Ser Asn Ser Glu Asp Lys Asp Arg Lys Gly Met Leu Gln Leu Lys Ile Ala Gly Ala Pro Glu Pro Leu Thr Val Thr Ala Pro Ser Leu Thr Ile Ala Glu Asn Met Ala Asp Leu Ile Asp Gly Tyr Cys Arg Leu Val Asn PC11699A.ST25.txt Gly Thr Ser Gln Ser Phe Ile Ile Arg Pro Gln Lys Glu Gly Glu Arg Ala Leu Pro Ser Ile Pro Lys Leu Ala Asn Ser Glu Lys Gln Gly Met Arg Thr His Ala Val Ser Val Ser Glu Thr Asp Asp Tyr Ala Glu Ile Ile Asp Glu Glu Asp Thr Tyr Thr Met Pro Ser Thr Arg Asp Tyr Glu Ile Gln Arg Glu Arg Ile Glu Leu Gly Arg Cys Ile Gly Glu Gly Gln Phe Gly Asp Val His Gln Gly Ile Tyr Met Ser Pro Glu Asn Pro Ala Leu Ala Val Ala Ile Arg Thr Cys Lys Asn Cys Thr Ser Asp Ser Val Arg Glu Lys Phe Leu Gln Glu Ala Leu Thr Met Arg Gln Phe Asp His Pro His Ile Val Lys Leu Ile Gly Val Ile Thr Glu Asn Pro Val Trp Ile Ile Met Glu Leu Cys Thr Leu Gly Glu Leu Arg Ser Phe Leu Gln Val Arg Lys Tyr Ser Leu Asp Leu Ala Ser Leu Ile Leu Tyr Ala Tyr Gln Leu Ser Thr Ala Leu Ala Tyr Leu Glu Ser Lys Arg Phe Val His Arg Asp Ile Ala Ala Arg Asn Val Leu Val Ser Ser Asn Asp Cys Val Lys Leu Gly Asp Phe Gly Leu Ser Arg Tyr Met Glu Asp Ser Thr Tyr Tyr Lys Ala Ser Lys Gly Lys Leu Pro Ile Lys Trp Met Ala Pro Glu PC11699A.ST25.txt Ser Ile Asn Phe Arg Arg Phe Thr Ser Ala Ser Asp Val Trp Met Phe Gly Val Cys Met Trp Glu Ile Leu Met His Gly Val Lys Pro Phe Gln Gly Val Lys Asn Asn Asp Val Ile Gly Arg Ile Glu Asn Gly Glu Arg Leu Pro Met Pro Pro Asn Cys Pro Pro Thr Leu Tyr Ser Leu Met Thr Lys Cys Trp Ala Tyr Asp Pro Ser Arg Arg Pro Arg Phe Thr Glu Leu Lys Ala Gln Leu Ser Thr Ile Leu Glu Glu Glu Lys Ala Gln Gln Glu Glu Arg Met Arg Met Glu Ser Arg Arg Gln Ala Thr Val Ser Trp Asp Ser Gly Gly Ser Asp Glu Ala Pro Pro Lys Pro Ser Arg Pro Gly Tyr Pro Ser Pro Arg Ser Ser Glu Gly Phe Tyr Pro Ser Pro Gln His Met Val Gln Thr Asn His Tyr Gln Val Ser Gly Tyr Pro Gly Ser His Gly Ile Thr Ala Met Ala Gly Ser Ile Tyr Pro Gly Gln Ala Ser Leu Leu Asp Gln Thr Asp Ser Trp Asn His Arg Pro Gln Glu Ile Ala Met Trp Gln Pro Asn Val Glu Asp Ser Thr Val Leu Asp Leu Arg Gly Ile Gly Gln Val Leu Pro Thr His Leu Met Glu Glu Arg Leu Ile Arg Gln Gln PC11699A.ST25.txt Gln Glu Met Glu Glu Asp Gln Arg Trp Leu Glu Lys Glu Glu Arg Phe Leu Lys Pro Asp Val Arg Leu Ser Arg Gly Ser Ile Asp Arg Glu Asp Gly Ser Leu Gln Gly Pro Ile Gly Asn Gln His Ile Tyr Gln Pro Val Gly Lys Pro Asp Pro Ala Ala Pro Pro Lys Lys Pro Pro Arg Pro Gly Ala Pro Gly His Leu Gly Ser Leu Ala Ser Leu Ser Ser Pro Ala Asp Ser Tyr Asn Glu Gly Val Lys Leu Gln Pro Gln Glu Ile Ser Pro Pro Pro Thr Ala Asn Leu Asp Arg Ser Asn Asp Lys Val Tyr Glu Asn Val Thr Gly Leu Val Lys Ala Val Ile Glu Met Ser Ser Lys Ile Gln Pro Ala Pro Pro Glu Glu Tyr Val Pro Met Val Lys Glu Val Gly Leu Ala Leu Arg Thr Leu Leu Ala Thr Val Asp Glu Thr Ile Pro Leu Leu Pro Ala Ser Thr His Arg Glu Ile Glu Met Ala Gln Lys Leu Leu Asn Ser Asp Leu Gly Glu Leu Ile Asn Lys Met Lys Leu Ala Gln Gln Tyr Val Met Thr Ser Leu Gln Gln Glu Tyr Lys Lys Gln Met Leu Thr Ala Ala His Ala Leu Ala Val Asp Ala Lys Asn Leu Leu Asp Val Ile Asp Gln Ala Arg Leu Lys Met Leu Gly Gln Thr Arg Pro His PC11699A.ST25.txt <210> 4 <211> 366 <212> PRT
<213> Human FRNK (amino acid 694-1052 of wild type FAK, including initiator M;
V5 epitope and His tag not included) <400> 4 Met Asp Tyr Pro Tyr Asp Val Glu Ser Arg Arg Gln Ala Thr Val Ser Trp Asp Ser Gly Gly Ser Asp Glu Ala Pro Pro Lys Pro Ser Arg Pro Gly Tyr Pro Ser Pro Arg Ser Ser Glu Gly Phe Tyr Pro Ser Pro Gln His Met Val Gln Thr Asn His Tyr Gln Val Ser Gly Tyr Pro Gly Ser His Gly Ile Thr Ala Met Ala Gly Ser Ile Tyr Pro Gly Gln Ala Ser Leu Leu Asp Gln Thr Asp Ser Trp Asn His Arg Pro Gln Glu Ile Ala Met Trp Gln Pro Asn Val Glu Asp Ser Thr Val Leu Asp Leu Arg Gly Ile Gly Gln Val Leu Pro Thr His Leu Met Glu Glu Arg Leu Ile Arg Gln Gln Gln Glu Met Glu Glu Asp Gln Arg Trp Leu Glu Lys Glu Glu Arg Phe Leu Lys Pro Asp Val Arg Leu Ser Arg Gly Ser Ile Asp Arg Glu Asp Gly Ser Leu Gln Gly Pro Ile Gly Asn Gln His Ile Tyr Gln Pro Val Gly Lys Pro Asp Pro Ala Ala Pro Pro Lys Lys Pro Pro Arg Pro Gly Ala Pro Gly His Leu Gly Ser Leu Ala Ser Leu Ser Ser Pro PC11699A.ST25.txt Ala Asp Ser Tyr Asn Glu Gly Val Lys Leu Gln Pro Gln Glu Ile Ser Pro Pro Pro Thr Ala Asn Leu Asp Arg Ser Asn Asp Lys Val Tyr Glu Asn Val Thr Gly Leu Val Lys Ala Val Ile Glu Met Ser Ser Lys Ile Gln Pro Ala Pro Pro Glu Glu Tyr Val Pro Met Val Lys Glu Val Gly Leu Ala Leu Arg Thr Leu Leu Ala Thr Val Asp Glu Thr Ile Pro Leu Leu Pro Ala Ser Thr His Arg Glu Ile Glu Met Ala Gln Lys Leu Leu Asn Ser Asp Leu Gly Glu Leu Ile Asn Lys Met Lys Leu Ala Gln Gln Tyr Val Met Thr Ser Leu Gln Gln Glu Tyr Lys Lys Gln Met Leu Thr Ala Ala His Ala Leu Ala Val Asp Ala Lys Asn Leu Leu Asp Val Ile-Asp Gln Ala Arg Leu ~Lys Met Leu Gly Gln Thr Arg Pro His <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 ggcagctgct120 taccttgacc ccaacttgaa tcacacacca aattcgagta ctaagactca cctgggtact180 ggtatggaac gttctcctgg tgcaatggag cgagtattaa aggtctttca ttattttgaa240 agcaatagtg agccaaccac ctgggccagt attatcaggc atggagatgc tactgatgtc300 aggggcatca ttcagaagat agtggacagt cacaaagtaa agcatgtggc ctgctatgga360 ttccgcctca gtcacctgcg PC11699A.ST25.txt 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 actggca.atc 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 PC11699A.ST25.txt 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 PC11699A.ST25.txt 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 PC11699A.ST25.txt 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 (pGenelFAKK454R/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 PC11699A.ST25.txt 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 PC11699A.ST25.txt 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> Human FAK mutant called FRNK (pGene/FRNK/V5-His) <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 PC11699A.ST25.txt atggaatcac agccatggct ggcagcatct atccaggtca300 ggcatctctt ttggaccaaa cagattcatg gaatcataga cctcaggaga tagcaatgtg360 gcagcccaat gtggaggact ctacagtatt ggacctgcga gggattgggc aagtgttgcc420 aacccatctg atggaagagc gtctaatccg acagcaacag gaaatggaag aagatcagcg ctggctggaa aaagaggaaa 480 gatttctgaa acctgatgtg agactctctc gaggcagtat540 tgacagggag gatggaagtc ttcagggtcc gattggaaac caacatatat atcagcctgt600 gggtaaacca gatcctgcag ctccaccaaa gaaaccgcct cgccctggag ctcccggtca660 tctgggaagc cttgccagcc tcagcagccc tgctgacagc tacaacgagg gtgtcaagct tcagccccag gaaatcagcc 720 cccctcctac tgccaacctg gaccggtcga atgataaggt780 gtacgagaat gtgacgggcc tggtgaaagc tgtcatcgag atgtccagta aaatccagcc840 agccccacca gaggagtatg tccctatggt gaaggaagtc ggcttggccc tgaggacatt900 attggccact gtggatgaga ccattcccct cctaccagcc agcacccacc gagagattga980 gatggcacag aagctattga actctgacct gggtgagctc atcaacaaga tgaaactggc1020 ccagcagtat gtcatgacca gcctccagca agagtacaaa aagcaaatgc tgactgctgc1080 tcacgccctg gctgtggatg ccaaaaactt actcgatgtc attgaccaag caagactgaa1140 aatgcttggg cagacgagac cacactccct gggcccttcg aaggtaagcc fiatccctaac cctctcctcg gtctcgattc 1200 tacgcgtacc ggtcatcatc accatcacca ttga <210> 9 <211> 27 <212> DNA
<213> FAKS'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

What is claimed is:

1. A method for identifying cell-active inhibitors of focal adhesion kinase (FAK) comprising:
(a) adding an inducing agent to mammalian cells to induce the expression of a gene encoding FAK, wherein said mammalian cells are stably transfected with said gene, and said gene is expressed in the presence of said inducing agent;
(b) adding a test compound;
(c) capturing the expressed FAK using a FAK capture agent; and (d) detecting phosphorylation of said FAK.

2. The method of claim 1 wherein said mammalian cells are coated on a first solid phase, and wherein phosphorylation of FAK is detected by exposing phosphorylated FAK to an anti-phospho-tyrosine antibody and detecting the presence of said antibody.

3. The method of claim 1 wherein said FAK capture agent comprises one or more types of antibodies.

4. The method of claim 3, wherein said one or more types of antibodies comprise an anti-phospho-tyrosine antibody.

5. The method of claim 1 wherein said phosphorylation is proportional to the binding of anti-phospho-tyrosine antibody to the captured FAK.

6. The method of claim 2 wherein said cells naturally adhere to the first solid phase and wherein said cells are lysed with a lysis buffer prior to capturing the expressed FAK.

7. The method of claim 1 wherein the inducing agent is an agonist for the expression of the gene encoding FAK.

8. The method of claim 1 wherein said test compound inhibits the kinase-dependent phosphorylation of FAK.

9. A method for measuring the cytotoxicity of a test compound comprising:
(a) stably transfecting mammalian cells with a gene encoding FAK, wherein said gene is expressed in the presence of an inducing agent;
(b) adding an inducing agent to induce the expression of said gene encoding FAK;
(c) adding the test compound;
(d) adding a cytotoxicity indicator to said cells; and, (e) detecting the cytoxicity of the test compound.

10. A mammalian cell stably transfected with a recombinant nucleic acid molecule, that encodes a protein comprising a sequence selected from the group consisting of SEQ ID NOS: 1, 2, 3 and 4, and wherein expression of said protein requires the presence of an inducing agent.

11. A mammalian cell that is stably transfected with a recombinant nucleic acid that inducibly expresses FAK protein.

12. The mammalian cell of claim 11, wherein the nucleic acid encodes proteins selected from the group consisting of human FAK splice variants, catalytic domain of human FAK, mouse FAK, rat FAK and chicken FAK.

13. A mammalian cell stably transfected with a recombinant nucleic acid molecule comprising a polynucleotide selected from the group consisting of SEQ
ID NOS. 5, 6, 7 and 8, and wherein expression of said polynucleotide requires the presence of an inducing agent.

14. A method for identifying cell-active inhibitors of focal adhesion kinase (FAK) comprising the steps of:
(a) coating a first solid phase with a homogeneous population of mammalian cells so that the cells adhere to the first solid phase, wherein said cells are stably transfected with a gene encoding FAK, and wherein said gene is expressed in the presence of an inducing agent;
(b) adding an inducing agent to induce the expression of said gene encoding FAK;
(c) adding a test compound;
(d) solubilizing the adhering cells to release the cell lysate;
(e) coating a second solid phase with a FAK capture agent so that the FAK
capture agent adheres to the second solid phase;
(f) exposing the cell lysate to the adhered FAK capture agent so that the FAK
capture agent captures FAK;
(g) exposing the captured FAK to an anti-phospho-tyrosine antibody ; and, (h) measuring binding of the anti-phospho-tyrosine antibody to the captured FAK, wherein the amount of anti-phospho-tyrosine antibody binding to the captured FAK is proportional to the amount of phosphorylation of said FAK.