WO2002085404A1 - Lkb1-binding proteins - Google Patents

Abstract

The invention relates to a new protein that binds to LKB1, a protein associated with Peutz-Jeghers Syndrome, and suppresses NFλB's transcriptional activity.

Description

LKB1-BINDING PROTEINS

RELATED APPLICATION

This application claims the benefit of United States Provisional Patent Application No. 60/281,503, filed April 4, 2001, the content of which is hereby incorporated by reference.

BACKGROUND

Peutz-Jeghers syndrome (PJS) is an autosomal dominant inherited disease characterized by hamartomatous polyps in the gastrointestinal tract and by mucocutaneous melanin pigmentation (Tomlinson et al., J. Med. Genet. 34: 1007-1011, 1997). It has been estimated that PJS patients are at least 10 times more likely to develop cancer than that of the general population (Giardiello et al, N. Engl. J. Med. 316:1511-1514, 1987). A great deal of attention has, therefore, been drawn to the study of the genetic bases of this syndrome, and to understand the molecular mechanisms of tumorigenesis in PJS. Recently, a locus for PJS was mapped to human chromosome 19pl3 by comparative genomic hybridization analysis of multiple hamartomas derived from a single PJS patient and by genetic linkage analysis. Use of polymorphic markers in the region showed that the presumptive wild-type alleles were lost in the hamartomas, which led to the suggestion that the target of the deletion was a PJS tumor suppressor gene (Hemminki et al., Nat. Genet. 15:87-90, 1997). Sequence analysis of the gene mutated in PJS patients suggested that it was a serine/threonine protein ldnases gene named LKBl (Hemminki et al, Nature 391:184-187, 1998; and Jenne et al, Nat. Genet. 18:38-43, 1998). LKBl has been described as a protein kinase highly expressed in human fetal liver and testis, as a nuclear protein kinase, and as homologous to an early embryonic protein kinase found in the cytoplasm of oocytes and eggs in Xenopus (Su et al. , J. Biol. Chem. 271:14430- 14437, 1996).

Although LKBl is implicated as a tumor suppressor gene in PJS, only a limited number of mutations in the coding region of LKBl have been identified in sporadic tumors of afflicted patients (Avizienyte et al, Cancer Res. 58:2087-2090, 1998; Bignell et al, Cancer Res. 58:1384-1386, 1998; and Resta et al, Cancer Res. 58:4799-4801, 1998). On the other hand, mutant proteins derived from PJS families exhibit a loss of LKBl kinase activity (Esteller et al., Oncogene 19:164-168, 2000; and Mehenni et al., Am. J. Hum. Genet.63:1641-1650, 1998). In addition, many of the mutations in LKBl found in PJS patients are predicted to disrupt or abolish the kinase activity of the protein. A functional and specific growth-suppressive activity of LKBl kinase has been demonstrated in several tumor cell lines that exhibit severely impaired LKBl kinase activity (Tiainen et al, Proc. Natl. Acad. Sci USA 96:9248-9251, 1999).

SUMMARY

The invention is based on the discovery of a human fetal liver protein that specifically binds to LK l. It was also discovered that this human protein suppresses NFκB-mediated transcriptional activation. Thus, the human protein to which this invention pertains has been designated fetal liver LKBl -interacting protein (FLIP). The full-length human FLIP cDNA is shown below:

TCGACTCTCCGACAACTCCTCAGAGGCGGATGTTTGGGGCCACTGGAATAAGCCCAGCACCAGCTGGGGTGTGGA -301 CAGCCTCGGGGGCCAGGCCAACTCTCCCGGACGGGTGCGGACGGCAGGGGCGGGGCCGGGCTGTAGGCGGTGGGA -226

GGGGTGGGGCCGGGCGAGAAGGGGCTGGGCGGGCTGCCGTCGGGCGGGGCTGCGCGGGGAAGGGCGGCGCTGCGC -151

GGGGGGCGGGGCTGCGTGGGGGAGGGCGGGGCCGGGCTGTTGTGGGTGGGCGGGGCGCACCGGGGAAAGTTCCGG -76

GAAGGGCGGCAGCCGGCGGGGCCCGGGCGCGGAAGTTGCCGGCGGCCGCGCGGCCTCCCGGGCGGGCCTACAGCC -1

ATGTCCCGGGACCCGGGGTCGGGCGGCTGGGAGGAGGCCCCGCGCGCAGCTGCCGCGCTCTGCACCCTGTACCAC 75 1 M S R D P G S G G W E E A P R A A A A C T L Y H

GAGGCCGGACAGCGGCTGCGCCGCCTGCAGGACCAGCTCGCTGCCCGCGACGCCCTCATCGCTCGCCTCCGCGCC 150 26 E A G Q R L R R L Q D Q L A A R D A L I A R L R A

CGCCTGGCCGCGCTGGAGGGGGACGCCGCGCCGTCCCTAGTGGACGCGCTGCTGGAGCAGGTTGCGCGCTTCCGG 225 51 R L A A E G D A A P S V D A L L E Q V A R F R

GAGCAGCTGCGAAGGCAGGAGGGCGGCGCCGCCGAGGCCCAGATGCGCCAGGAAATTGAGAGGCTGACTGAGCGA 300 76 E Q L R R Q E G G A A E A Q M R Q E I E R L T E R

CTAGAAGAAAAAGAGAGGGAGATGCAGCAGCTGCTGAGCCAGCCCCAACACGAGCGAGAGAAGGAAGTCGTCCTG 375 101 E E K E R E M Q Q S Q P Q H E R E K E V V

CTACGGAGGAGCATGGCAGAAGGGGAGCGCGCCCGGGCCGCCAGTGACGTCCTGTGCCGCTCCTTGGCCAACGAG 450 126 R R Ξ M A E G E R A R A A S D V L C R S L A N E

ACCCATCAGCTGCGGAGGACGCTGACCGCCACCGCCCACATGTGTCAGCATCTGGCCAAGTGTCTGGATGAACGA 525 151 T H Q R R T L T A T A H M C Q H L A K C L D E R

CAGCATGCACAAAGGAATGTGGGGGAGAGAAGTCCTGACCAGTCGGAACACACAGATGGGCACACCTCTGTCCAG 600 176 Q H A Q R N V G E R S P D Q S E H T D G H T S V Q

AGTGTTATTGAGAAGTTGCAGGAAGAAAATCGACTGTTAAAACAGAAGGTGACTCACGTTGAAGACCTCAATGCC 675 201 S V I E K L Q E E N R L K Q K V T H V E D N A

AAGTGGCAGCGCTACAACGCCAGCAGGGACGAATACGTGAGGGGGCTCCATGCGCAGCTCAGGGGGCTGCAGATC 750 226 K W Q R Y N A S R D E Y V R G L H A Q L R G L Q I

CCCCACGAGCCCGAGCTGATGAGGAAGGAGATCTCCCGGCTCAACAGACAGTTGGAAGAGAAAATAAATGACTGT 825 251 P H E P E M R K E I S R L N R Q E E K I N D C

GCCGAAGTGAAGCAGGAGCTGGCGGCCTCCAGGACGGCCCGGGATGCTGCGTTGGAGCGGGTGCAGATGCTGGAA 900 276 A E V K Q E L A A S R T A R D A A L E R V Q M L E

CAGCAGATTCTCGCTTACAAGGATGACTTCATGTCAGAAAGGGCCGATCGGGAACGGGCTCAAAGTAGGATTCAA 975 301 Q Q I A Y K D D F M S E R A D R E R A Q S R I Q GAACTGGAGGAAAAGGTCGCCTCTTTGCTGCACCAGGTGTCCTGGAGACAGGATTCTCGAGAGCCAGACGCCGGC 98 326 E L E E K V A S L H Q V S W R Q D S R E P D A G

CGGATTCACGCTGGGAGCAAAACTGCCAAGTATTTGGCCGCCGACGCATTAGAGCTTATGGTGCCTGGTGGCTGG 1050 351 R I H A G S K T A K Y L A A D A E M V P G G W

AGGCCTGGGACTGGGTCCCAGCAGCCAGAACCCCCTGCAGAGGGCGGGCATCCTGGCGCGGCCCAGAGAGGCCAG 1125 376 R P G T G S Q Q P E P P A E G G H P G A A Q R G Q

GGGGACCTTCAGTGCCCTCACTGCCTGCAGTGCTTCAGTGACGAGCAAGGGGAAGAGCTCCTCAGGCATGTGGCC 1200 401 G D L Q C P H C Q C F S D E Q G E E L R H V A

GAGTGCTGCCAGTGACCGAGACTCACCCGTGCCCTTGCGGCCTCCTGGCCCGGTGCAGCTGCCCTCAGGGACAGG 1275 426 E c C Q * (SEQ ID NO: 2)

GTGGGTGCTCTCAGATGCCATGGGTTGAGCTCTACTGAGAGCCAAGGCCCCTAGAATAGTTGCGGGGCACTCTGA 1350

TCGTTCACTTTGGTCCCTTTGGCTATGGAACAGGCTGGGTCACAGGGAACTGCCAGTGAGGCTGGAGGCTGGAGG 1425

TGGAGATGGGGTCAGGAACATCTGGCAGAGGGAGGTCCCAGTCTGTGTCTCCATCAGGCTTAAGCCAGAGCTATC 1500 TGGTGCTGGTGTGCCAGCCCCTCCCCCAGCCTGCCTAGAAAGGGGTGGCTGCCTGAGGGAGTCACTTGTATGGTC 1575

CCCAGGGTGGGAGCCCCATCCTGTTCTATGGAATAAAGCGTCGCCTCTCTGCCTCGAACCAGTCAAATGGAGTAT 1650 TGCGGCTGCACGTCA (SEQ ID NO : 3 )

The nucleotide sequence encoding the human FLIP protein (i.e., from the ATG start codon to the codon immediately before the stop codon in SEQ ID NO:3) is designated SEQ ID NO:l.

Accordingly, the invention features a pure polypeptide that includes an amino acid sequence at least 70% (e.g., at least 75, 80, 85, 90, 95, 98, or 100%) identical to SEQ ID NO:2. Once expressed in a cell, the polypeptide binds to LKBl and represses the transcriptional activity of NFKB, as determined by method shown in the examples below or other applicable methods.

A "pure polypeptide" is a polypeptide free from other biological macromolecules, e.g., it is at least 75% (e.g., at least 80, 85, 95, or 99%) pure by dry weight. Purity can be measured by any appropriate standard method, for example, by column chromatography, polyacrylamide gel electrophoresis, or HPLC analysis.

The "percent identity" of two amino acid sequences is determined using the algorithm of Karlin and Altschul (Proc. Natl. Acad. Sci. USA 87:2264-2268, 1990), modified as in Karlin and Altschul (Proc. Natl. Acad. Sci. USA 90:5873-5877, 1993). Such an algorithm is incorporated into the NBLAST and XBLAST programs of Altschul et al. (J. Mol. Biol.215:403-410, 1990). BLAST nucleotide searches are performed with the NBLAST program, score = 100, wordlength = 12. BLAST protein searches are performed with the XBLAST program, score = 50, wordlength = 3. Where gaps exist between two sequences, Gapped BLAST is utilized as described in Altschul et al. (Nucleic Acids Res.25:3389-3402, 1997). When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) are used. See http://www.ncbi.nlm.nih.gov.

The invention also features an isolated nucleic acid encoding a polypeptide of the invention, and the complement of the nucleic acid. An example of a nucleic acid within the invention is an isolated nucleic acid that hybridizes under stringent conditions (i.e., hybridization at 65°C, 0.5X SSC, followed by washing at 45°C, 0.1X SSC) to SEQ ID NO:l, or the complement of SEQ ID NO:l. Such a nucleic acid can have at least 10 (e.g., at least 20, 30, 50, 100, 200, 500, or 1000) nucleotides in length. The nucleic acids can be used as primers in PCR-based detection methods or as probes in nucleic acid blots (e.g., Northern blots) for diagnostic purpose (see below).

An "isolated nucleic acid" is a nucleic acid the structure of which is not identical to that of any naturally occurring nucleic acid or to that of any fragment of a naturally occurring genomic nucleic acid spanning more than three separate genes. The term therefore covers, for example, (a) a DNA which has the sequence of part of a naturally occurring genomic DNA molecule but is not flanked by both of the coding sequences that flank that part of the molecule in the genome of the organism in which it naturally occurs; (b) a nucleic acid incorporated into a vector or into the genomic DNA of a prokaryote or eukaryote in a manner such that the resulting molecule is not identical to any naturally occurring vector or genomic DNA; (c) a separate molecule such as a cDNA, a genomic fragment, a fragment produced by polymerase chain reaction (PCR), or a restriction fragment; and (d) a recombinant nucleotide sequence that is part of a hybrid gene, i.e., a gene encoding a fusion protein. Specifically excluded from this definition are nucleic acids present in mixtures of different (i) DNA molecules, (ii) transfected cells, or (iii) cell clones, e.g., as these occur in a DNA library such as a cDNA or genomic DNA library. In addition, the invention features a method of (1) expressing in a cell a transcript, i.e., transcript I, that hybridizes under above-described high stringency conditions to SEQ ID NO:l, or (2) expressing in a cell a transcript, i.e., transcript π, that is complementary to transcript I. Transcript I, when expressed in a cell, can serve as an anti-sense RNA that binds to endogenous FLIP mRNA to prevent it from being translated into a functional protein. Therefore, this method can be used in gene therapy for treating diseases associated with over-expression of the FLIP gene. Transcript II may encode a FLIP protein, and when expressed in a cell, is translated into a FLIP protein. Thus, this method can be used for production of a polypeptide of the invention, or for treating a patient having a disease associated with insufficient FLIP gene expression.

The invention further features a diagnostic method for determining whether a patient has a disorder (e.g., PJS) associated with abnormal expression of the FLIP gene by comparing the level of FLIP gene expression in a test sample from the patient (e.g., a biopsy or a body fluid) with the level of FLIP gene expression in a control sample from a normal person. A higher FLIP gene expression level in the test sample indicates that the patient has a disorder associated with over-expression of the FLIP gene. A lower FLIP gene expression level in the test sample indicates that the patient has a disorder associated with insufficient expression of the FLIP gene. The FLIP gene expression level can be determined by measuring the amount of a FLIP mRNA (e.g., using the nucleic acids of this invention; see above) or the amount of a FLIP protein (e.g., using an antibody raised against a polypeptide of this invention).

The invention also features a method of identifying a compound that alters interaction between FLIP and LKBl. The method includes quantifying the extent of binding of FLIP to LKBl in the presence of a compound and comparing it to the extent of binding of FLIP to LKB 1 in the absence of the compound. A lower extent of binding in the presence of the compound indicates that the compound disrupts the interaction between FLIP and LKB 1. A higher extent of binding in the presence of the compound indicates that the compound enhances the interaction between FLIP and LKBl. This method can be carried out both in vivo (i.e., using nucleic acids encoding FLIP and LKBl) and in vitro (i.e., using purified FLIP and LKBl proteins). Compounds thus identified are candidates for treating diseases associated with abnormal interaction between FLIP and LKBl. Also within the scope of the invention is a method of identifying a compound that alters suppression of the transcriptional activity of NFKB by FLIP. The method includes contacting FLIP with a cell expressing NFKB and quantifying the transcriptional activity of NFKB in the presence of a compound. If the transcriptional activity in the presence of the compound is lower than in the absence of the compound, it indicates that the compound enhances the suppression of the transcriptional activity of NFKB by FLIP. If the transcriptional activity is higher in the presence of the compound than in the absence of the compound, it indicates that the compound reduces the suppression of the transcriptional activity of NFKB by FLIP. Compounds thus identified are candidates for treating diseases associated with abnormal suppression of the transcriptional activity of NFKB by FLIP.

The details of one or more embodiments of the invention are set forth in the accompanying description below. Other advantages, features, and objects of the invention will be apparent from the detailed description, and from the claims.

DETAILED DESCRIPTION

The invention relates to new FLIP proteins and nucleic acids encoding them that specifically bind to LKBl, the protein associated with PJS. In fact, mutations that knock out LKBl kinase activity, which appears to be the case in PJS patients, also knock out the binding of LKB 1 to FLIP. It is therefore hypothesized that a FLIP activity or lack thereof is a cause of PJS downstream from LKBl. Uses The nucleic acid molecules, proteins, protein homologues, and antibodies described herein can be used in one or more of the following methods: a) screening assays; b) predictive medicine (e.g., diagnostic assays, prognostic assays, monitoring clinical trials, and pharmacogenetics); and c) methods of treatment (e.g., therapeutic and prophylactic). The isolated nucleic acid molecules of the invention can be used, for example, to express a FLIP protein (e.g., via a recombinant expression vector in a host cell in gene therapy applications), to detect a FLIP mRNA (e.g., in a biological sample) or a genetic alteration in a FLIP gene, and to modulate FLIP activity. The FLIP proteins can be used to treat disorders characterized by insufficient or excessive production of a FLIP substrate or production of FLIP inhibitors. In addition, the FLIP proteins can be used to screen for naturally occurring FLIP substrates, to screen for drugs or compounds which modulate FLIP activity, as well as to treat disorders characterized by insufficient or excessive production of FLIP protein or production of FLIP protein forms that have decreased, aberrant, or unwanted activity compared to FLIP wild type protein (e.g., in liver cancer). Moreover, the anti-FLIP antibodies of the invention can be used to detect and isolate FLIP proteins, regulate the bioavailability of FLIP proteins, and modulate FLIP activity.

A method of evaluating a compound for the ability to interact with, e.g., bind, a subject FLIP polypeptide is provided. The method includes: contacting the compound with the subject FLIP polypeptide; and evaluating ability of the compound to interact with, e.g., to bind or form a complex with, the subject FLIP polypeptide. This method can be performed in vitro, e.g., in a cell free system, or in vivo, e.g., in a two-hybrid interaction trap assay. This method can be used to identify naturally occurring molecules that interact with subject FLIP polypeptide. It can also be used to find natural or synthetic inhibitors of a subject FLIP polypeptide. Screening Assays

The invention provides methods (also referred to herein as "screening assays") for identifying modulators, i.e., candidate or test compounds or agents (e.g., proteins, peptides, peptidomimetics, peptoids, small molecules, or other drugs) which bind to FLIP proteins, have a stimulatory or inhibitory effect on, for example, FLIP expression or FLIP activity, or have a stimulatory or inhibitory effect on, for example, the expression or activity of a FLIP substrate. Compounds thus identified can be used to modulate the activity of target gene products (e.g., FLIP genes) in a therapeutic protocol, to elaborate the biological function of the target gene product, or to identify compounds that disrupt normal target gene interactions.

In one embodiment, the invention provides assays for screening candidate or test compounds which are substrates of a FLIP protein or polypeptide or a biologically active portion thereof. In another embodiment, the invention provides assays for screening candidate or test compounds which bind to or modulate the activity of a FLIP protein or polypeptide or a biologically active portion thereof.

The test compounds of the present invention can be obtained using any of the numerous approaches in combinatorial library methods known in the art, including: biological libraries; peptoid libraries (libraries of molecules having the functionalities of peptides, but with a novel, non-peptide backbone which is resistant to enzymatic degradation but which nevertheless remains bioactive; see, e.g., Zuckermann et al, J. Med. Chem. 37:2678-85, 1994); spatially addressable parallel solid phase or solution ^' phase libraries; synthetic library methods requiring deconvolution; the "one-bead one- compound" library method; and synthetic library methods using affinity chromatography selection. The biological library and peptoid library approaches are limited to peptide libraries, while the other four approaches are applicable to peptide, non-peptide oligomer, or small molecule libraries of compounds (Lam, Anticancer Drug Des. 12: 145, 1997).

Examples of methods for the synthesis of molecular libraries can be found in the art, for example in DeWitt et al, Proc. Natl. Acad. Sci. U.S.A. 90:6909, 1993; Erb et al, Proc. Natl. Acad. Sci. USA 91:11422, 1994; Zuckermann et al, J. Med. Chem. 37:2678, 1994; Cho et al, Science 261:1303, 1993; Carrell et al, Angew. Chem. Int. Ed. Engl. 33:2059, 1994; Carell et al, Angew. Chem. Int. Ed. Engl. 33:2061, 1994; and in Gallop et al, J. Med. Chem. 37:1233, 1994.

Libraries of compounds may be presented in solution (e.g., Houghten, BioTechniques 13:412-421, 1992), on beads (Lam, Nature 354:82-84, 1991), chips (Fodor, Nature 364:555-556, 1993), bacteria (Ladner, USP 5,223,409), spores (Ladner USP '409), plasmids (Cull et al. Proc Natl Acad Sci USA 89:1865-1869, 1992), or on phage (Scott and Smith, Science 249:386-390, 1990; Devlin, Science 249:404-406, 1990; Cwirla et al, Proc. Natl. Acad. Sci. 87:6378-6382, 1990; Felici, J. Mol. Biol. 222:301- 310, 1991; Ladner supra.). In one embodiment, an assay is a cell-based assay in which a cell which expresses a FLIP protein or biologically active portion thereof is contacted with a test compound, and the ability of the test compound to modulate FLIP activity is determined. Determining the ability of the test compound to modulate FLIP activity can be accomplished by monitoring, for example, binding to LKBl. The cell, for example, can be of mammalian origin, e.g., human.

The ability of the test compound to modulate FLIP binding to a compound, e.g., LKBl, or to bind to FLIP can also be evaluated. This can be accomplished, for example, by coupling the compound, e.g., the substrate, with a radioisotope or enzymatic label such that binding of the compound, e.g., the substrate, to FLIP can be determined by detecting the labeled compound, e.g., substrate, in a complex. Alternatively, FLIP could be coupled with a radioisotope or enzymatic label to monitor the ability of a test compound to modulate FLIP binding to a FLIP substrate in a complex. For example, compounds (e.g., FLIP substrates) can be labeled with ¹²⁵I, ³⁵S, ¹⁴C, or ³H, either directly or indirectly, and the radioisotope detected by direct counting of radioemmission or by scintillation counting. Alternatively, compounds can be enzymatically labeled with, for example, horseradish peroxidase, alkaline phosphatase, or luciferase, and the enzymatic label detected by determination of conversion of an appropriate substrate to product. The ability of a compound (e.g., a FLIP substrate) to interact with FLIP with or without the labeling of any of the interactants can be evaluated. For example, a microphysiometer can be used to detect the interaction of a compound with FLIP without the labeling of either the compound or the FLIP (McConnell et al, Science 257:1906- 1912, 1992). As used herein, a "microphysiometer" (e.g., Cytosensor) is an analytical instrument that measures the rate at which a cell acidifies its environment using a light- addressable potentiometric sensor (LAPS). Changes in this acidification rate can be used as an indicator of the interaction between a compound and FLIP.

In yet another embodiment, a cell-free assay is provided in which a FLIP protein or biologically active portion thereof is contacted with a test compound and the ability of the test compound to bind to the FLIP protein or biologically active portion thereof is evaluated. Preferred biologically active portions of the FLIP proteins to be used in assays of the present invention include fragments which participate in interactions with non-FLIP molecules, e.g., fragments with high surface probability scores. Soluble and/or membrane-bound forms of isolated proteins (e.g., FLIP proteins or biologically active portions thereof) can be used in the cell-free assays of the invention. When membrane-bound forms of the protein are used, it may be desirable to utilize a solubilizing agent. Examples of such solubilizing agents include non-ionic detergents such as n-octylglucoside, n-dodecylglucoside, n-dodecylmaltoside, octanoyl-N- methylglucamide, decanoyl-N-methylglucamide, Triton® X-100, Triton® X-114, Thesit®, Isotridecypoly(ethylene glycol ether)_n, 3-[(3-cholan_idopropyl)dimethylamminio]-l- propane sulfonate (CHAPS), 3-[(3-cholarmdopropyl)dimethylamminio]-2-hydroxy-l- propane sulfonate (CHAPSO), or N-dodecyl-N,N-dimethyl-3-ammonio-l-propane sulfonate. Cell-free assays involve preparing a reaction mixture of the target gene protein and the test compound under conditions and for a time sufficient to allow the two components to interact and bind, thus forming a complex that can be removed and/or detected.

The interaction between two molecules can also be detected, e.g., using fluorescence energy transfer (FET) (see, for example, U.S. Patent Nos. 5,631,169 and 4,868,103). A fluorophore label on the first, "donor" molecule is selected such that its emitted fluorescent energy will be absorbed by a fluorescent label on a second, "acceptor" molecule, which in turn is able to fluoresce due to the absorbed energy. Alternately, the "donor" protein molecule may simply utilize the natural fluorescent energy of tryptophan residues. Labels are chosen that emit different wavelengths of light, such that the "acceptor" molecule label may be differentiated from that of the "donor." Since the efficiency of energy transfer between the labels is related to the distance separating the molecules, the spatial relationship between the molecules can be assessed. In a situation in which binding occurs between the molecules, the fluorescent emission of the "acceptor" molecule label in the assay should be maximal. An FET binding event can be conveniently measured through standard fluorometric detection means well known in the art (e.g., using a fluorimeter). In another embodiment, determining the ability of the FLIP protein to bind to a target molecule can be accomplished using real-time Biomolecular Interaction Analysis (BIA) (see, e.g., Sjolander and Urbaniczky, Anal. Chem. 63:2338-2345, 1991 and Szabo et al, Curr. Opin. Struct. Biol. 5:699-705, 1995). "Surface plasmon resonance" or "BIA" detects biospecific interactions in real time, without labeling any of the interactants (e.g., BIAcore). Changes in the mass at the binding surface (indicative of a binding event) result in alterations of the refractive index of light near the surface (the optical phenomenon of surface plasmon resonance (SPR)), resulting in a detectable signal which can be used as an indication of real-time reactions between biological molecules.

In one embodiment, the target gene product or the test substance is anchored onto a solid phase. The target gene product/test compound complexes anchored on the solid phase can be detected at the end of the reaction. Preferably, the target gene product can be anchored onto a solid surface, and the test compound, (which is not anchored), can be labeled, either directly or indirectly, with detectable labels discussed herein.

It may be desirable to immobilize either FLIP, an anti-FLIP antibody or its target molecule to facilitate separation of complexed from uncomplexed forms of one or both of the proteins, as well as to accommodate automation of the assay. Binding of a test compound to a FLIP protein, or interaction of a FLIP protein with a target molecule in the presence and absence of a candidate compound, can be accomplished in any vessel suitable for containing the reactants. Examples of such vessels include microtiter plates, test tubes, and micro-centrifuge tubes. In one embodiment, a fusion protein can be provided which adds a domain that allows one or both of the proteins to be bound to a matrix. For example, glutathione-S-transferase/FLDP fusion proteins or glutathione-S- transferase/ target fusion proteins can be adsorbed onto glutathione sepharose beads (Sigma Chemical, St. Louis, MO) or glutathione derivatized microtiter plates, which are then combined with the test compound or the test compound and either the non-adsorbed target protein or FLIP protein, and the mixture incubated under conditions conducive to complex formation (e.g., at physiological conditions for salt and pH). Following incubation, the beads or microtiter plate wells are washed to remove any unbound components, the matrix immobilized in the case of beads. Complexes are determined either directly or indirectly, for example, as described above. Alternatively, the complexes can be dissociated from the matrix, and the level of FLIP binding or activity determined using standard techniques.

Other techniques for immobilizing either a FLIP protein or a target molecule on matrices include using conjugation of biotin and streptavidin. Biotinylated FLIP protein or target molecules can be prepared from biotin-NHS (N-hydroxy-succinimide) using techniques known in the art (e.g., biotinylation kit, Pierce Chemicals, Rockford, EL), and immobilized in the wells of streptavidin-coated 96 well plates (Pierce Chemical).

In order to conduct the assay, the non-immobilized component is added to the coated surface containing the anchored component. After the reaction is complete, unreacted components are removed (e.g., by washing) under conditions such that any complexes formed will remain immobilized on the solid surface. The detection of complexes anchored on the solid surface can be accomplished in a number of ways. Where the previously non-immobilized component is pre-labeled, the detection of label immobilized on the surface indicates that complexes were formed. Where the previously non-immobilized component is not pre-labeled, an indirect label can be used to detect complexes anchored on the surface; e.g., using a labeled antibody specific for the immobilized component (the antibody, in turn, can be directly labeled or indirectly labeled with, e.g., a labeled anti-Ig antibody).

In one embodiment, this assay is performed utilizing antibodies reactive with FLIP protein or target molecules but which do not interfere with binding of the FLIP protein to its target molecule. Such antibodies can be derivatized to the wells of the plate, and unbound target or FLIP protein trapped in the wells by antibody conjugation. Methods for detecting such complexes, in addition to those described above for the GST-immobilized complexes, include immunodetection of complexes using antibodies reactive with the FLIP protein or target molecule, as well as enzyme-linked assays which rely on detecting an enzymatic activity associated with the FLIP protein or target molecule.

Alternatively, cell free assays can be conducted in a liquid phase. In such an assay, the reaction products are separated from unreacted components by any of a number of standard techniques including but not limited to differential centrifugation (see, for example, Rivas and Minton, Trends Biochem Sci 18:284-7, 1993); chromatography (gel filtration chromatography, ion-exchange chromatography); electrophoresis (see, e.g., Ausubel et al, eds. Current Protocols in Molecular Biology 1999, J. Wiley: New York.); and immunoprecipitation (see, for example, Ausubel et al, eds. (1999) Current Protocols in Molecular Biology, J. Wiley: New York). Such resins and chromatographic techniques are known to one skilled in the art (see, e.g., Heegaard, J Mol Recognit 11:141-8, 1998; Hage and Tweed, J Chromatogr B Biomed Sci Appl. 699:499-525, 1997). Further, fluorescence energy transfer may also be conveniently utilized, as described herein, to detect binding without further purification of the complex from solution. In a preferred embodiment, the assay includes contacting the FLIP protein or biologically active portion thereof with a known compound (e.g., LKBl) which binds FLIP to form an assay mixture, contacting the assay mixture with a test compound, and determining the ability of the test compound to interact with a FLIP protein, where determining the ability of the test compound to interact with a FLIP protein includes determining the ability of the test compound to preferentially bind to FLIP or biologically active portion thereof, or to modulate the activity of a target molecule, as compared to the known compound.

The target gene products of the invention can, in vivo, interact with one or more cellular or extracellular macromolecules, such as proteins. For the purposes of this discussion, such cellular and extracellular macromolecules are referred to herein as

"binding partners." Compounds that disrupt such interactions can be useful in regulating the activity of the target gene product. Such compounds can include, but are not limited to molecules such as antibodies, peptides, and small molecules. The preferred target genes/products for use in this embodiment are the FLIP genes herein identified. In an alternative embodiment, the invention provides methods for determining the ability of the test compound to modulate the activity of a FLIP protein through modulation of the activity of a downstream effector of a FLIP target molecule. For example, the activity of the effector molecule on an appropriate target can be determined, or the binding of the effector to an appropriate target can be determined, as previously described.

To identify compounds that interfere with the interaction between the target gene product and its cellular or extracellular binding partner(s), a reaction mixture containing the target gene product and the binding partner is prepared, under conditions and for a time sufficient, to allow the two products to form a complex. In order to test an inhibitory agent, the reaction mixture is provided in the presence and absence of the test compound. The test compound can be initially included in the reaction mixture, or can be added at a time subsequent to the addition of the target gene and its cellular or extracellular binding partner. Control reaction mixtures are incubated without the test compound or with a placebo. The formation of any complexes between the target gene product and the cellular or extracellular binding partner is then detected. The formation of a complex in the control reaction, but not in the reaction mixture containing the test compound, indicates that the compound interferes with the interaction of the target gene product and the interactive binding partner. Additionally, complex formation within reaction mixtures containing the test compound and normal target gene product can also be compared to complex formation within reaction mixtures containing the test compound and mutant target gene product. This comparison can be important in those cases where it is desirable to identify compounds that disrupt interactions of mutant but not normal target gene products.

These assays can be conducted in a heterogeneous or homogeneous format. Heterogeneous assays involve anchoring either the target gene product or the binding partner onto a solid phase, and detecting complexes anchored on the solid phase at the end of the reaction. In homogeneous assays, the entire reaction is carried out in a liquid phase. In either approach, the order of addition of reactants can be varied to obtain different information about the compounds being tested. For example, test compounds that interfere with the interaction between the target gene products and the binding partners, e.g., by competition, can be identified by conducting the reaction in the presence of the test substance. Alternatively, test compounds that disrupt preformed complexes, e.g., compounds with higher binding constants that displace one of the components from the complex, can be tested by adding the test compound to the reaction mixture after complexes have been formed. The various formats are briefly described below. In a heterogeneous assay system, either the target gene product or the interactive cellular or extracellular binding partner is anchored onto a solid surface (e.g., a microtiter plate), while the non-anchored species is labeled either directly or indirectly. The anchored species can be immobilized by non-covalent or covalent attachments. Alternatively, an immobilized antibody specific for the species to be anchored can be used to anchor the species to the solid surface.

In order to conduct the assay, the partner of the immobilized species is exposed to the coated surface with or without the test compound. After the reaction is complete, unreacted components are removed (e.g., by washing) and any complexes formed will remain immobilized on the solid surface. Where the non-immobilized species is pre- labeled, the detection of label immobilized on the surface indicates that complexes were formed. Where the non-immobilized species is not pre-labeled, an indirect label can be used to detect complexes anchored on the surface, e.g., using a labeled antibody specific for the initially non-immobilized species. The antibody, in turn, can be directly labeled or indirectly labeled with, e.g., a labeled anti-Ig antibody. Depending upon the order of addition of reaction components, test compounds that inhibit complex formation or that disrupt preformed complexes can be detected.

Alternatively, the reaction can be conducted in a liquid phase in the presence or absence of the test compound, the reaction products separated from unreacted components, and complexes detected, e.g., using an immobilized antibody specific for one of the binding components to anchor any complexes formed in solution and a labeled antibody specific for the other partner to detect anchored complexes. Again, depending upon the order of addition of reactants to the liquid phase, test compounds that inhibit complex formation or that disrupt preformed complexes can be identified. In an alternate embodiment, a homogeneous assay can be used. For example, a preformed complex of the target gene product and the interactive cellular or extracellular binding partner product is prepared so that either the target gene products or their binding partners are labeled, but the signal generated by the label is quenched due to complex formation (see, e.g., U.S. Patent No. 4,109,496 that utilizes this approach for immunoassays). The addition of a test substance that competes with and displaces one of the species from the preformed complex will result in the generation of a signal above background. In this way, test substances that disrupt target gene product-binding partner interaction can be identified.

In yet another aspect, the FLIP proteins can be used as "bait proteins" in a two- hybrid assay or three-hybrid assay (see, e.g., U.S. Patent No. 5,283,317; Zervos et al, Cell 72:223-232, 1993; Madura et al, J. Biol. Chem. 268:12046-12054, 1993; Bartel et al, BioTechniques 14:920-924, 1993; Iwabuchi et al, Oncogene 8:1693-1696, 1993; and Brent WO94/10300) to identify other proteins, which bind to or interact with FLIP ("FLIP-binding proteins" or "FLIP-bp") and are involved in FLIP activity. Such FLIP-bps can be activators or inhibitors of signals by the FLIP proteins or FLIP targets as, for example, downstream elements of a FLIP-mediated signaling pathway. The two-hybrid system is based on the modular nature of most transcription factors, which consist of separable DNA-binding and activation domains. Briefly, the assay utilizes two different DNA constructs. In one construct, the gene that codes for a FLIP protein is fused to a gene encoding the DNA binding domain of a known transcription factor (e.g., GAL-4). In the other construct, a DNA sequence, from a library of DNA sequences, that encodes an unidentified protein ("prey" or "sample") is fused to a gene that codes for the activation domain of the known transcription factor. (Alternatively, the FLIP protein can be fused to the activator domain.) If the "bait" and the "prey" proteins are able to interact, in vivo, forming a FLEP-dependent complex, the DNA- binding and activation domains of the transcription factor are brought into close proximity. This proximity allows transcription of a reporter gene (e.g., lacZ) which is operably linked to a transcriptional regulatory site responsive to the transcription factor. Expression of the reporter gene can be detected and cell colonies containing the functional transcription factor can be isolated and used to obtain the cloned gene which encodes the protein that interacts with the FLIP protein. In another embodiment, modulators of FLIP expression are identified. For example, a cell or cell free mixture is contacted with a candidate compound and the expression of FLIP mRNA or protein evaluated relative to the level of expression of FLIP mRNA or protein in the absence of the candidate compound. When expression of FLIP mRNA or protein is greater in the presence of the candidate compound than in its absence, the candidate compound is identified as a stimulator of FLIP mRNA or protein expression. Alternatively, when expression of FLIP mRNA or protein is less (statistically significantly less) in the presence of the candidate compound than in its absence, the candidate compound is identified as an inhibitor of FLIP mRNA or protein expression. The level of FLIP mRNA or protein expression can be determined by methods described herein for detecting FLIP mRNA or protein.

, In another aspect, the invention pertains to a combination of two or more of the assays described herein. For example, a modulating agent can be identified using a cell- based or a cell free assay, and the ability of the agent to modulate the activity of a FLIP protein can be confirmed in vivo, e.g., in an animal such as an animal model for hepatocellular carcinoma.

This invention further pertains to novel agents identified by the above-described screening assays. Accordingly, it is within the scope of this invention to further use an agent identified as described herein (e.g., a FLIP modulating agent, an antisense FLIP nucleic acid molecule, a FLIP-specific antibody, or a FLIP-binding partner) in an appropriate animal model to determine the efficacy, toxicity, side effects, or mechanism of action, of treatment with such an agent. Furthermore, novel agents identified by the above-described screening assays can be used for treating cancers, e.g., liver cancer. Use of FLIP Molecules as Surrogate Markers

The FLIP molecules of the invention are also useful as markers of disorders or disease states, as markers for precursors of disease states, as markers for predisposition of disease states, as markers of drug activity, or as markers of the pharmacogenomic profile of a subject. Using the methods described herein, the presence, absence and or quantity of the FLIP molecules of the invention may be detected, and may be correlated with one or more biological states in vivo. For example, the FLIP molecules of the invention may serve as surrogate markers for one or more disorders or disease states or for conditions leading up to disease states. As used herein, a "surrogate marker" is an objective biochemical marker which correlates with the absence or presence of a disease or disorder, or with the progression of a disease or disorder (e.g., with the presence or absence of a liver tumor). The presence or quantity of such markers is independent of the disease. Therefore, these markers may serve to indicate whether a particular course of treatment is effective in lessening a disease state or disorder. Surrogate markers are of particular use when the presence or extent of a disease state or disorder is difficult to assess through standard methodologies (e.g., early stage tumors), or when an assessment of disease progression is desired before a potentially dangerous clinical endpoint is reached (e.g., an assessment of cardiovascular disease may be made using cholesterol levels as a surrogate marker, and an analysis of HIN infection may be made using HIN RΝA levels as a surrogate marker, well in advance of the undesirable clinical outcomes of myocardial infarction or fully-developed AIDS). Examples of the use of surrogate markers in the art include those described in Koomen et al, J. Mass. Spectrom. 35:258-264, 2000; and James, AIDS Treatment News Archive 209, 1994.

The FLIP molecules of the invention are also useful as pharmacodynamic markers. As used herein, a "pharmacodynamic marker" is an objective biochemical marker which correlates specifically with drug effects. The presence or quantity of a pharmacodynamic marker is not related to the disease state or disorder for which the drug is being administered; therefore, the presence or quantity of the marker is indicative of the presence or activity of the drug in a subject. For example, a pharmacodynamic marker may be indicative of the concentration of the drug in a biological tissue, in that the marker is either expressed or transcribed or not expressed or transcribed in that tissue in relationship to the level of the drug. In this fashion, the distribution or uptake of the drug may be monitored by the pharmacodynamic marker. Similarly, the presence or quantity of the pharmacodynamic marker may be related to the presence or quantity of the metabolic product of a drug, such that the presence or quantity of the marker is indicative of the relative breakdown rate of the drug in vivo. Pharmacodynamic markers are of particular use in increasing the sensitivity of detection of drug effects, particularly when the drug is administered in low doses. Since even a small amount of a drug may be sufficient to activate multiple rounds of marker (e.g., a FLIP marker) transcription or expression, the amplified marker may be in a quantity which is more readily detectable than the drug itself. Also, the marker may be more easily detected due to the nature of the marker itself; for example, using the methods described herein, anti-FLIP antibodies may be employed in an immune-based detection system for a FLIP protein marker, or FLIP-specific radiolabeled probes may be used to detect a FLIP mRNA marker. Furthermore, the use of a pharmacodynamic marker may offer mechanism-based prediction of risk due to drug treatment beyond the range of possible direct observations. Examples of the use of pharmacodynamic markers in the art are described in Matsuda et al. US 6,033,862; Hattis et al, Env. Health Perspect. 90:229-238, 1991; Schentag, Am. J. Health-Syst. Pharm.' 56 Suppl. 3:S21-S24, 1999; and Nicolau, Am. J. Health-Syst. Pharm. 56 Suppl. 3:S16-S20, 1999.

The FLIP molecules of the invention are also useful as pharmacogenomic markers. As used herein, a "pharmacogenomic marker" is an objective biochemical marker which correlates with a specific clinical drug response or susceptibility in a subject (see, e.g., McLeod et al., Eur. J. Cancer 35: 1650-1652, 1999). The presence or quantity of the pharmacogenomic marker is related to the predicted response of the subject to a specific drug or class of drugs prior to administration of the drug. By assessing the presence or quantity of one or more pharmacogenomic markers in a subject, a drug therapy which is most appropriate for the subject, or which is predicted to have a greater degree of success, may be selected. For example, based on the presence or quantity of RNA, or protein (e.g., FLIP protein or RNA) for specific tumor markers in a subject, a drug or course of treatment may be selected which is optimized for the treatment of the specific tumor likely to be present in the subject. Similarly, the presence or absence of a specific sequence mutation in FLIP DNA may correlate with FLIP drug response. The use of pharmacogenomic markers therefore permits the application of the most appropriate treatment for each subject without having to administer the therapy. Pharmacogenomics

The FLIP molecules of the present invention, as well as agents, or modulators which have a stimulatory or inhibitory effect on FLIP activity (e.g., FLIP gene expression) as identified by a screening assay described herein can be administered to individuals to treat (prophylactically or therapeutically) FLIP associated disorders (e.g., liver cancer or PJS) associated with aberrant or unwanted FLIP activity. In conjunction with such treatment, pharmacogenomics (i.e., the study of the relationship between an individual's genotype and that individual's response to a foreign compound or drug) may be considered. Differences in metabolism of therapeutics can lead to severe toxicity or therapeutic failure by altering the relation between dose and blood concentration of the pharmacologically active drug. Thus, a physician or clinician may consider applying knowledge obtained in relevant pharmacogenomics studies in determining whether to administer a FLIP molecule or FLIP modulator as well as tailoring the dosage and/or therapeutic regimen of treatment with a FLIP molecule or FLIP modulator.

Pharmacogenomics deals with clinically significant hereditary variations in the response to drugs due to altered drug disposition and abnormal action in affected persons. See, for example, Eichelbaum et al, Clin. Exp. Pharmacol. Physiol. 23:983-985, 1996 and Linder et al, Clin. Chem. 43:254-266, 1997. In general, two types of pharmacogenetic conditions can be differentiated. Genetic conditions transmitted as a single factor altering the way drugs act on the body (altered drug action) or genetic conditions transmitted as single factors altering the way the body acts on drugs (altered drug metabolism). These pharmacogenetic conditions can occur either as rare genetic defects or as naturally- occurring polymorphisms. For example, glucose-6-phosphate dehydrogenase deficiency (G6PD) is a common inherited enzymopathy in which the main clinical complication is haemolysis after ingestion of oxidant drugs (anti-malarials, sulfonamides, analgesics, nitrofurans) and consumption of fava beans.

One pharmacogenomics approach to identifying genes that predict drug response, known as "a genome- wide association," relies primarily on a high-resolution map of the human genome consisting of already known gene-related markers (e.g., a "bi-allelic" gene marker map which consists of 60,000-100,000 polymorphic or variable sites on the human genome, each of which has two variants). Such a high-resolution genetic map can be compared to a map of the genome of each of a statistically significant number of patients taking part in a Phase π/ HI drug trial to identify markers associated with a particular observed drug response or side effect. Alternatively, such a high resolution map can be generated from a combination of some ten-million known single nucleotide polymorphisms (SNPs) in the human genome. As used herein, a "SNP" is a common alteration that occurs in a single nucleotide base in a stretch of DNA. For example, a SNP may occur once per every 1000 bases of DNA. A SNP may be involved in a disease process, however, the vast majority may not be disease-associated. Given a genetic map based on the occurrence of such SNPs, individuals can be grouped into genetic categories depending on a particular pattern of SNPs in their individual genome. In such a manner, treatment regimens can be tailored to groups of genetically similar individuals, taking into account traits that may be common among such genetically similar individuals.

Alternatively, a method termed the "candidate gene approach", can be utilized to identify genes that predict drug response. According to this method, if a gene that encodes a drug's target is known (e.g., a FLIP protein of the present invention), all common variants of that gene can be fairly easily identified in the population, and it can be determined if having one version of the gene versus another is associated with a particular drug response.

Alternatively, a method termed the "gene expression profiling", can be utilized to identify genes that predict drug response. For example, the gene expression of an animal dosed with a drug (e.g., a FLIP molecule or FLIP modulator of the present invention) can give an indication whether gene pathways related to toxicity have been turned on. Information generated from more than one of the above pharmacogenomics approaches can be used to determine appropriate dosage and treatment regimens for prophylactic or therapeutic treatment of an individual. This knowledge, when applied to dosing or drug selection, can avoid adverse reactions or therapeutic failure and thus enhance therapeutic or prophylactic efficiency when treating a subject with a FLIP molecule or FLIP modulator, such as a modulator identified by one of the exemplary screening assays described herein.

The present invention further provides methods for identifying new agents, or combinations, that are based on identifying agents that modulate the activity of one or more of the gene products encoded by one or more of the FLIP genes of the present invention, where these products may be associated with resistance of the cells to a therapeutic agent. Specifically, the activity of the proteins encoded by the FLIP genes of the present invention can be used as a basis for identifying agents for overcoming agent resistance. By blocking the activity of one or more of the resistance proteins, target cells, e.g., human cells, will become sensitive to treatment with an agent that the unmodified target cells were resistant to.

Monitoring the influence of agents (e.g., drugs) on the expression or activity of a FLIP protein can be applied in clinical trials. For example, the effectiveness of an agent determined by a screening assay as described herein to increase FLIP gene expression, protein levels, a FLIP activity can be monitored in clinical trials of subjects exhibiting decreased FLIP gene expression, protein levels, or FLIP activity. Alternatively, the effectiveness of an agent determined by a screening assay to decrease FLIP gene expression, protein levels, or a FLIP activity can be monitored in clinical trials of subjects exhibiting increased FLIP gene expression, protein levels, or FLIP activity. In such clinical trials, the expression or activity of a FLIP gene, and preferably other genes that have been implicated in, for example, a FLIP-associated disorder can be used as a "read out" or marker of the phenotype of a particular cell. The specific examples below are to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way whatsoever. Without further elaboration, it is believed that one skilled in the art can, based on the description herein, utilize the present invention to its fullest extent. All publications recited herein are hereby incorporated by reference in their entirety.

Materials and Methods cDNA synthesis. cDNA was synthesized from the human fetal liver poly(A)⁺ RNA (Clontech) using Superscript RT reverse transcriptase in the Superscript Plasmid System according to the manufacturer's instructions (Life Technologies, Inc.). The entire coding region of LKBl was first produced by PCR amplification using the oligonucleotides 5'- GGG TCC AGC ATG GAG GTG GTG GAC CCG CAG-3' (SEQ ID NO:4) and 5'-CGG CCA GCC TCA CTG CTG CTT GCA GGC CGA-3' (SEQ ID NO:5), with the fetal liver cDNA as template. DNA sequencing was used to confirm the PCR product. Yeast two-hybrid screening. The yeast GAL4 two-hybrid system was described previously in Chevray et al, Proc. Natl. Acad. Sci. USA 89:5789-5793, 1992. The entire coding region of LKBl cDNA was fused in frame with the GAL4 DNA-binding domain into the pPC62 vector to form pPC62-LKBl, and transformed into Saccharomyces cerevisiae yeast strain YPB2. The human fetal liver cDNA library was fused to the GAL4 activation domain on the plasmid pPC86, and used to screen for the LKBl -interacting partners. Potential positive yeast colonies were first selected on agar plates lacking histidine over a period of 7 days. His⁺ colonies were then streaked out on selective plates in the presence of 10 mM 3-aminotriazole and checked for β-galactosidase activity by using a colony-lift filter procedure to confirm the positive interaction. Plasmids were recovered from positive colonies and subjected to further analysis.

Northern blot hybridization. Human MTN multiple human tissue RNA blots (Clontech) were hybridized with a cDNA probe generated by PCR, which contained the entire coding region of FLIP. The 1.0 kb probe was purified, labeled with α-³²P-dCTP, and hybridized to the RNA blots using established protocols. A probe of glyceraldehyde-3- phosphate dehydrogenase clone included in the kit was used as a control in the hybridization.

Plasmid construction. Mammalian expression vectors, pFLAG-CMV-2 and pEGFP-Nl, were obtained from Sigma and Clontech, respectively.

Three deletion mutants of LKBl, LKBl- NΔ87, LKBl- NΔ135, and LKBl- CΔ46, were constructed by restriction enzyme digestion of the original plasmid. Specifically, the entire coding sequence of LKBl on the plasmid pBluescript II SK+ (Stratagene) was digested with each of BamHI, Sphl, and Smal and another enzyme corresponding to the polycloning sites on the vector to remove the N-terminal 87, the N-terminal 135, or the C- terminal 46 amino acid coding sequences. The digested DNA fragments were then subcloned through several bacterial vectors to manipulate appropriate restriction enzyme cloning sites and ligated into the yeast two-hybrid, bacterial, and mammalian expression vectors. LKB1-K78M and SL26 were created by site-directed mutagenesis in a PCR protocol. The oligonucleotide 5'-CTT CTT GAG GAT CaT GAC GGC CCT CCT-3' (SEQ ID NO:6) was used to introduce a lysine to methionine change at the residue 78. The lower-case letter in the primer sequence indicate the introduced mutation site. On the other hand, the oligonucleotide 5'-TCT CCA TCC GGC AGA AC A GCT GGT TCC GGA-3' (SEQ ID NO:7) was used to form a 9 bp deletion mutation of LKB 1-SL26. The LKBl mutant of SL29 was constructed by a two-step cloning procedure. The first was a PCR reaction to amplify the partial LKBl fragments flanking the truncated site of SL29 mutation, followed by standard restriction enzyme digestion and ligation.

For bacterial recombinant protein expression, pGST-LKBl and pGST-FLEP were constructed by subcloning the original DNA amplified by PCR into pGEX4T-2

(Pharmacia Biotech Inc.). For the thioredoxin-tagged constructs of LKBl and FLIP, DNA fragments were ligated into the plasmid pThioHisC (Invitrogen) to form pTRX-LKB 1 and pTRX-FLEP, respectively. The bacterial expression constructs were transformed into E. coli strain BL21(DE3) (Novagen) for protein expression. The induction and purification of the recombinant proteins were performed following the individual manufacturer's recommendations .

Kinase assay. Protein kinase assay was performed as described previously in Su et al, J. Biol. Chem. 271:14430-14437, 1996. A sample of recombinant LKBl proteins or immunoprecipitates was incubated in kinase buffer at 30°C for 20 minutes in the presence of Mn²⁺ and γ-³²P-ATP. The reaction was then terminated and subjected to SDS-PAGE and autoradiography.

Cell culture and transfection. 293T human embryonic kidney cells were maintained in DMEM medium (Life Technologies, Inc.) supplemented with 10% fetal calf serum. Transfection of 293T cells was performed using calcium phosphate precipitation. Antibodies. LKBl antiserum was raised in rabbits, and FLIP antiserum was raised in mice against purified recombinant LKBl and FLIP proteins, respectively. The antibody was affinity-purified on nitrocellulose blots of recombinant proteins as described in Tang et al, Meth. Cell Biol. 37:95-104, 1993. The secondary antibodies, rhodamine-conjugated donkey anti-rabbit IgG and FITC-conjugated goat anti-mouse IgG, were purchased from Jackson ImmunoResearch. Two anti-FLAG monoclonal antibodies (M5 and M2) were obtained from Sigma, and a polyclonal anti-FLAG antibody of rabbit (D8) was purchased from Santa Cruz Biotechnology.

Immunoprecipitation. Immunoprecipitation was performed essentially as described in Su et al, J. Biol. Chem. 271:14430-14437, 1996. Human 293T cells were transfected with either pFLAG-CMN-LKBl or pEGFP-FLIP. Cell lysates were prepared 24 hours after transfection in a lysis buffer (50 mM Hepes, pH 7.0, 0.5% Νonidet P-40, 250 mM ΝaCl, 1 mM phenylmethylsulfonyl fluoride, 5 mM EDTA, 5 mM EGTA, 2 μg/ml each of aprotinin, pepstatin, and leupeptin, 0.5 mM sodium orthovanadate, 5 mM sodium fluoride, and 50 mM β-glycerophosphate). Each of the cell lysates were incubated with either monoclonal antibody to FLAG or polyclonal antibody to FLIP, and immobilized onto protein A/G-Sepharose (Pharmacia Biotechnology). Immunoprecipitates were resolved by SDS-PAGE, followed by immunoblotting with either anti-FLIP or anti-FLAG. The detection of antigen-antibody interaction was based on enhanced chemiluminescence.

In vitro pull-down assay. Cell lysates were prepared from 293T cells transfected with either pFLAG-CMN-LKBl or empty vector as a control, precleared with GST- glutathione-agarose beads, incubated with soluble GST-FLIP protein, and mixed with reduced glutathione-agarose beads (Sigma). The protein-bound agarose beads were then collected and washed, followed by SDS-PAGE analysis. The FLIP-bound protein was detected by immunoblotting with anti-FLAG.

Immunofluorescence microscopy. Immunofluorescent microscopy was performed as described in Ghosh et al, J. Biol. Chem. 275:7184-7188. Briefly, transfected 293T cells were seeded on glass coverslips coated with polyamino acids. The slides were placed in DMEM containing 10% fetal calf serum, and the cells were allowed to grow for 2-3 days until they reached a semi-confluent state. The cultured cells were washed and fixed with 2% formaldehyde in PBS. The fixed cells were first incubated with primary antibodies, washed twice in PBS, and reacted with fluorophore-conjugated secondary antibodies. The cells were washed again with PBS, analyzed under immunofluorescence microscope, and photographed. NFκB-dependent reporter gene assay. For the reporter gene assay, 293T cells (1.6 x 10⁵ cells/well) were seeded into six-well (35 mm) plates. At 24 hours after seeding, cells were transfected with an NFκB-dependent luciferase reporter construct (Stratagene) and FLIP and LKBl expression constructs. A plasmid containing the β-galactosidase gene under the control of the β-actin promoter (pAct-β-Gal) was used for normalizing transfection efficiency. Luciferase activity was measured with a luciferase assay kit (Roche Molecular Biochemicals) using Monolight 2010 (Analytical Luminescence Laboratory).

Results Cloning and characterization of human LKBl. To investigate the function of

LKBl kinase, the LKBl cDNA was first cloned from human fetal liver. The human fetal liver was previously shown to be an abundant source of the LKB 1 transcripts. The primers were designed according to GenBank accession number U63333 and used in a PCR reaction to amplify human LKB 1 from a pool of cDNA prepared from human fetal liver. DNA sequencing analysis confirmed that the PCR product of 1.3 kilobases contained the entire coding region of the human LKBl clone. Athioredoxin (TRX)-fusion LKBl was constructed and expressed in bacterial cells. The purified TRX-LKB1 was tested for its kinase activity and shown to autophosphorylate in vitro. This result indicated that the cloned LKBl was functional. In addition, like the Xenopus XEEK1 kinase, LKBl had a preference for Mn²⁺ over Mg²⁺. Both the human LKB 1 kinase and the Xenopus XEEK1 kinase showed very similar biochemical characteristics.

Cloning of FLIP by yeast two-hybrid screening. The yeast GAL4 system was used to perform two-hybrid screening for protein-protein interactions. The screening was performed in a two-step selection in which two reporter genes, HIS3⁺ and LacZ, were used. A fusion protein containing the GAL4 DNA binding domain and the entire coding region of LKBl sequence was used as bait in the screening of a human fetal liver cDNA library, which was cloned into the GAL4 activation domain. After an initial screening of around 50,000 colonies, several His⁺ colonies were isolated. Plasmids from these His⁺ colonies were recovered and subjected to further analysis. Only one of these colonies later proved to be a true positive clone by re-transformation of recovered plasmids into the yeast strain and by β-galactosidase assay. The DNA insert from this positive clone was isolated and sequenced. The sequence of 1.7-kilobase insert cDNA contained an open reading frame of 1287 bp, which encodes a protein of 429 amino acid residues (see above). Northern analysis of mRNA from various human tissues using cloned cDNA as a probe identified a unique mRNA species of -1.8 kilobases, indicating that the cDNA analyzed was close to full-length. This clone was designated FLIP, as explained above. Finally, given the high homology between the human LKBl and the Xenopus XEEK1, e.g. 86% identity in protein sequence, Xenopus XEEK1 was also able to interact with FLIP in the yeast two-hybrid analysis. On the other hand, several unrelated mammalian protein kinase clones failed to interact with FLIP under the same experimental conditions. Thus the interaction between LKBl and FLIP is specific.

Characterization of the FLIP clone. The protein sequence of FLIP did not resemble any known protein sequence. The full-length FLIP gene encodes 429 amino acid residues with a predicted molecular weight of 47 kDa. After in vitro translation, FLIP migrated at the 47 kDa region of a gel, an electrophoretic mobility similar to that of the predicted molecular weight. Using a computer server program CoilScan (SeqWeb, Genetics Computer Group, Inc.) to analyze the structure of protein sequence, four presumptive coiled coil segments in FLIP were located.

Northern blot analysis was performed to define the tissue expression pattern of FLIP. Multiple tissue blots revealed a widespread major FLIP mRNA of -1.8 kilobase, which was present at different levels depending on the tissue. This result suggested that the expression of FLIP was ubiquitous among various human tissues. The expression level of the FLIP transcript in brain was either very low or non-detectable, whereas expression was slightly elevated in testis and colon. The actual expression level of FLIP on the tissue blots was normalized based on a control probed for glyceralaldehyde-3- phosphate dehydrogenase (G3PDH) on the same blots.

Whether FLIP could act as a substrate of the LKBl kinase was next examined. Both GST-tagged FLIP and TRX-tagged LKBl were expressed and purified separately to near homogeneity in bacterial cells. It was observed that the TRX-tagged LKB 1 construct yielded much more soluble protein than that of GST-tagged construct in bacteria. Soluble GST-FLIP and TRX-LKB1 proteins were mixed and incubated in a kinase buffer containing either Mg²⁺ or Mn²⁺ and radiolabeled ATP. The reaction was subjected to SDS- PAGE and autoradiography. No phosphorylation on GST-FLIP was detected in spite of strong autophosphorylation in TRX-LKB1. To demonstrate this assay in a more physiological context, FLIP and Flag-LKBl mammalian expression vectors were constructed and co-transfected into 293T cells. Co-immunoprecipitation was performed using either anti-FLIP antibody or anti-Flag antibody, and the immunoprecipitate was subjected to a kinase assay. No phosphorylation of FLIP was observed in either case. These results suggested that FLIP was probably not a substrate of the LKB 1 kinase.

In vitro protein-protein binding assay and co-immunoprecipitation. To test further for a physical interaction between FLIP and LKBl, an in vitro pull-down assay was performed. A GST fusion protein of FLIP was constructed and expressed in bacteria. GST-tagged FLIP was immobilized onto glutathione beads and incubated with Flag- LKBl -transfected cell lysates. After extensive washing, the protein bound to the beads was subjected to SDS-PAGE, followed by immunoblotting with an anti-Flag antibody. Unexpextedly, a specific band of Flag-LKBl was retained by the GST-FLIP glutathione beads. However, when GST glutathione beads or mock transfected cell lysates were used as negative controls, both controls failed to pull down the LKBl protein. These results suggested that FLIP was able to bind LKBl specifically.

The interaction between FLIP and LKBl was also tested in vivo by co-transfecting Flag-LKBl with either Flag-LKBl or FLIP-GFP constructs into 293T cells. Cell lysates were subjected to immunoprecipitation with anti-Flag monoclonal antibody (M5) that recognized the epitope-tag of LKB 1, analyzed by SDS-PAGE, and immunoblotted with anti-FLIP antibody. Unexpectedly, a 66 kDa FLIP-GFP band was detected in the immunoprecipitate from lysates of cells co-transfected with Flag-LKBl and FLIP-GFP, indicating the association of FLIP with LKBl in vivo. A lower band of 63 kDa, which was also present on the immunoblot, was most likely a variant form of FLIP-GFP generated by internal initiation of translation in the construct. The internal initiation products of 66 kDa and 63 kDa was confirmed by in vitro translation of the same construct. Immunoprecipitation with anti-FLIP antibody followed by immunoblotting with anti-Flag antibody also demonstrated association between FLIP and LKBl. The association between FLIP-GFP and Flag-LKBl was not seen in lysates of cells transfected with either construct alone. These results confirmed the interaction between FLIP and LKBl in vivo. Defining the interaction domains between LKBl and FLIP. To further characterize the interaction between LKB 1 and FLIP, a series of deletion mutants of LKB 1 and FLIP were generated by either PCR or restriction enzyme digestion. The truncated LKBl fragments were fused to the GAL4 DNA binding domain, and their ability to interact with the full-length FLIP fused to the GAL4 activation domain was examined in the yeast two- hybrid system. LKBl- NΔ87, which has a deletion of the N-terminal 87 amino acids, was still capable of interacting with FLIP. However, LKB 1- NΔ135 and LKB 1- CΔ46, which has a deletion of the N-terminal 135 amino acids or the C-terminal 46 amino acids, respectively, lost the ability to interact with FLIP. Therefore, two regions of the LKBl sequence, namely Domain I and II, were identified as being necessary for the interaction between FLIP and LKBl. Domain I and Domain II included the stretch of amino acid residues from about 88 to 135 and from about 387 to 433, respectively.

In addition, the epitope-tagged recombinant proteins were created and expressed in bacteria. The purified mutant proteins were assayed for kinase activity in vitro, and it was discovered that none of the mutant proteins retained the autophosphorylation activity. This result was consistent with the observation that all three truncations encompassed the kinase domain in LKB 1.

In another experiment, FLIP was truncated into two halves, each containing either the N-terminal 171 amino acids (CΔ169) or the C-terminal 169 amino acids (NΔ171), and each fused to the GAL4 activation domain. The truncated FLIP proteins were then tested for the ability to interact with the full-length LKB 1 in the two-hybrid system. Neither half of FLIP was able to interact with LKB 1.

Subcellular localization of FLIP. To characterize the cellular function of FLIP, the localization of expressed LKBl and FLIP was examined by indirect immunofluorescent microscopy. FLIP and LKBl mammalian expression constructs were prepared and transfected into 293T cells. Expressed proteins were detected using either a rabbit polyclonal antibody to LKBl or a mouse polyclonal antibody to FLIP. Immunofluorescent staining was performed 24 hours post-transfection using rhodamine-conjugated anti-rabbit IgG or FITC-conjugated anti-mouse IgG. Almost exclusive nuclear localization of LKBl in 293T cells was observed. In addition, the truncated form of LKBl (deletion of the first N-terminal 87 amino acids) appeared to be cytoplasmic. These results confirm that LKBl is a nuclear protein. Unexpectedly, using the same approach, it was found that FLIP was localized exclusively in the cytoplasm. FLIP suppresses the transcriptional activity of NFKB. In order to further explore the function of FLIP in mediating the tumor suppressor activity of LKB 1 , the effect of FLIP on the regulation of various gene promoters was examined. Unexpectedly, it was found that FLIP strongly suppresses expression from an NFKB -responsive reporter system in a dose-dependent manner in 293T cells.

OTHER EMBODIMENTS

All of the features disclosed in this specification may be combined in any combination. Each feature disclosed in this specification may be replaced by an alternative feature serving the same, equivalent, or similar purpose. Thus, unless expressly stated otherwise, each feature disclosed is only an example of a generic series of equivalent or similar features.

From the above description, one skilled in the art can easily ascertain the essential characteristics of the present invention, and without departing from the spirit and scope thereof, can make various changes and modifications of the invention to adapt it to various usages and conditions. Thus, other embodiments are also within the scope of the following claims.

Claims

WHAT IS CLAIMED IS:

1. A pure polypeptide comprising an amino acid sequence at least 70% identical to SEQ ID NO:2, wherein the polypeptide binds to LKBl.

2. The polypeptide of claim 1, wherein the amino acid sequence is at least 80% identical to SEQ ID NO:2.

3. The polypeptide of claim 2, wherein the amino acid sequence is at least 90% identical to SEQ ID NO:2.

4. The polypeptide of claim 3, wherein the amino acid sequence is SEQ ID NO:2.

5. An isolated nucleic acid encoding the polypeptide of claim 1, or a complementary sequence thereof.

6. An isolated nucleic acid encoding the polypeptide of claim 2, or a complementary sequence thereof.

7. An isolated nucleic acid encoding the polypeptide of claim 3, or a complementary sequence thereof.

8. An isolated nucleic acid encoding the polypeptide of claim 4, or a complementary sequence thereof.

9. An isolated nucleic acid that hybridizes under high stringency conditions to SEQ ID NO:l, or a complementary sequence thereof.

10. The nucleic acid of claim 9, wherein the nucleic acid has at least 10 nucleotides in length.

11. A method of expressing a transcript in a cell, the method comprising: introducing a vector into a cell, the vector containing a nucleic acid encoding a transcript; and expressing the transcript in the cell; wherein the transcript hybridizes under high stringency conditions to SEQ ID NO:l, or a complementary sequence thereof.

12. The method of claim 11, wherein the transcript encodes the polypeptide of claim 1.

13. The method of claim 11, wherein the transcript encodes the polypeptide of claim 2.

14. The method of claim 11, wherein the transcript encodes the polypeptide of claim 3.

15. The method of claim 11, wherein the transcript encodes the polypeptide of claim 4.

16. A method of detecting abnormal expression of an FLIP gene in a cell, the method comprising: providing a cell expressing an FLIP gene, and quantifying an expression level of the FLIP gene in the cell, wherein the expression level of the FLIP gene in the cell, if different from that in a normal cell, indicates abnormal expression of the FLIP gene.

17. The method of claim 16, wherein the expression level of the FLIP gene is quantified by measuring the amount of an FLIP mRNA in the cell.

18. The method of claim 16, wherein the expression level of the FLIP gene is quantified by measuring the amount of an FLIP protein in the cell.

19. A method of identifying a compound that alters interaction between FLIP and LKBl, the method comprising quantifying the extent of binding of FLIP to LKBl in the presence of a compound, wherein the extent of binding in the presence of the compound, if lower than in the absence of the compound, indicates that the compound disrupts the interaction between FLIP and LKB 1 ; and the extent of binding in the presence of the compound, if higher than in the absence of the compound, indicates that the compound enhances the interaction between FLIP and LKBl.

20. A method of identifying a compound that alters suppression of the transcriptional activity of NFKB by FLIP, the method comprising: contacting FLIP with a cell expressing NFKB, and quantifying the transcriptional activity of NFKB in the presence of a compound, wherein the transcriptional activity in the presence of the compound, if lower than in the absence of the compound, indicates that the compound enhances the suppression of the transcriptional activity of NFKB by FLIP; and the transcriptional activity in the presence of the compound, if higher than in the absence of the compound, indicates that the compound reduces the suppression of the transcriptional activity of NFKB by FLIP.