Nucleotide and protein sequences of lats genes and methods based thereon

Abstract

The present invention relates to a tumor suppressor gene, termed large tumor suppressor (lats). The invention provides nucleotide sequences of lats genes, and amino acid sequences of their encoded proteins, as well as derivatives (e.g., fragments) and analogs thereof. Diagnostic methods and pharmaceutical compositions are also provided. The invention further relates to treatment or prevention of disease or disorder involving cell proliferation by administering compounds that promote lats activity (e.g., lats, an agonist of lats; nucleic acids that encode lats).

What is claimed is:  
     
         1 . A purified lats protein.  
     
     
         2 . The protein of  claim 1  which is a human protein.  
     
     
         3 . The protein of  claim 1  which is a D. melanogaster protein.  
     
     
         4 . The protein of  claim 1  which is a mouse protein.  
     
     
         5 . The protein of  claim 1  which is a mammalian protein.  
     
     
         6 . The protein of  claim 2  which comprises the amino acid sequence substantially as set forth in SEQ ID NO:4.  
     
     
         7 . A purified protein encoded by a nucleic acid hybridizable to the lats DNA sequence in plasmid PBS(KS)-h-lats as deposited with the ATCC and assigned accession number 69769.  
     
     
         8 . A purified protein encoded by a nucleic acid hybridizable to a DNA having a sequence consisting of the coding region of SEQ ID NO:7.  
     
     
         9 . The protein of  claim 2  which is encoded by plasmid pBS(KS)-h-lats as deposited with the ATCC and assigned accession number 69769.  
     
     
         10 . A purified derivative or analog of the protein of  claim 1 , which displays one or more functional activities of a lats protein.  
     
     
         11 . The derivative or analog of  claim 10  which is able to be bound by an antibody directed against a lats protein.  
     
     
         12 . A purified fragment of a lats protein comprising a domain of the lats protein selected from the group consisting of a lats C-terminal domain 3 (LCD3), lats C-terminal domain 2 (LCD2), lats C-terminal domain 1 (LCD1), kinase domain, a kinase subdomain, lats flanking domain (LFD), lats split domain 1 (LSD1), lats split domain 2 (LSD2), SH3-binding domain, and opa repeat domain.  
     
     
         13 . A molecule comprising the fragment of  claim 12 .  
     
     
         14 . A protein comprising an amino acid sequence that has at least 60% identity to a domain of a lats protein, in which the percentage identity is determined over an amino acid sequence of identical size to the domain.  
     
     
         15 . A protein comprising an amino acid sequence that has at least 90% identity to a domain of a lats protein, in which the percentage identity is determined over an amino acid sequence of identical size to the domain.  
     
     
         16 . The derivative or analog of  claim 10 , which inhibits proliferation of a cell.  
     
     
         17 . A chimeric protein comprising a fragment of a lats protein consisting of at least 6 amino acids fused via a covalent bond to an amino acid sequence of a second protein, in which the second protein is not a lats protein.  
     
     
         18 . The chimeric protein of  claim 17  in which the fragment of a lats protein is a fragment capable of being bound by an anti-lats antibody.  
     
     
         19 . The fragment of  claim 12  which additionally lacks one or more domains of the lats protein.  
     
     
         20 . An antibody which is capable of binding a lats protein.  
     
     
         21 . The antibody of  claim 20  which is monoclonal.  
     
     
         22 . A molecule comprising a fragment of the antibody of  claim 21 , which fragment is capable of binding a lats protein.  
     
     
         23 . An isolated nucleic acid comprising a nucleotide sequence encoding a lats protein.  
     
     
         24 . The nucleic acid of  claim 23  which is a DNA.  
     
     
         25 . An isolated nucleic acid comprising a nucleotide sequence complementary to the nucleotide sequence of  claim 23 .  
     
     
         26 . The nucleic acid of  claim 23  in which the lats protein is a human lats protein.  
     
     
         27 . An isolated nucleic acid comprising the lats coding sequence contained in plasmid pBS(KS)-h-lats as deposited with the ATCC and assigned accession number 69769.  
     
     
         28 . An isolated nucleic acid hybridizable to the lats DNA sequence in plasmid pBS(KS)-h-lats as deposited with the ATCC and assigned accession number 69769.  
     
     
         29 . An isolated nucleic acid hybridizable to a DNA having a sequence consisting of the coding region of SEQ ID NO:7.  
     
     
         30 . An isolated nucleic acid comprising a fragment of a lats gene consisting of at least 8 nucleotides.  
     
     
         31 . An isolated nucleic acid comprising a nucleotide sequence encoding a fragment of a lats protein that displays one or more functional activities of the lats protein.  
     
     
         32 . An isolated nucleic acid comprising a nucleotide sequence encoding the chimeric protein of  claim 17 .  
     
     
         33 . An isolated nucleic acid comprising a nucleotide sequence encoding a protein, said protein comprising the amino acid sequence of SEQ ID NO:4.  
     
     
         34 . An isolated nucleic acid comprising a nucleotide sequence encoding the fragment of  claim 12 .  
     
     
         35 . An isolated nucleic acid comprising a nucleotide sequence encoding the protein of  claim 14 .  
     
     
         36 . A recombinant cell containing the nucleic acid of  claim 23 , in which the nucleotide sequence encoding the lats protein is under the control of a promoter that is not a native lats gene promoter.  
     
     
         37 . A recombinant cell containing a nucleic acid vector comprising the nucleic acid of  claim 26 .  
     
     
         38 . A recombinant cell containing the nucleic acid of  claim 34 .  
     
     
         39 . A recombinant cell containing the nucleic acid of  claim 35 .  
     
     
         40 . A method of producing a lats protein comprising growing a recombinant cell containing the nucleic acid of  claim 23 , in which the nucleotide sequence encoding the lats protein is under the control of a promoter that is not a native lats gene promoter, such that the encoded lats protein is expressed by the cell, and recovering the expressed lats protein.  
     
     
         41 . A method of producing a lats protein comprising growing a recombinant cell containing a nucleic acid vector comprising the nucleic acid of  claim 26  such that the encoded lats protein is expressed by the cell, and recovering the expressed lats protein.  
     
     
         42 . A method of producing a lats fragment comprising growing a recombinant cell containing the nucleic acid of  claim 34  such that the encoded lats fragment is expressed by the cell, and recovering the expressed lats fragment.  
     
     
         43 . A method of producing a protein comprising a fragment of a lats protein, which method comprises growing a recombinant cell containing the nucleic acid of  claim 35  such that the encoded protein is expressed by the cell, and recovering the expressed protein.  
     
     
         44 . The product of the process of  claim 40 .  
     
     
         45 . The product of the process of  claim 41 .  
     
     
         46 . The product of the process of  claim 42 .  
     
     
         47 . The product of the process of  claim 43 .  
     
     
         48 . A pharmaceutical composition comprising a therapeutically effective amount of a lats protein; and a pharmaceutically acceptable carrier.  
     
     
         49 . The composition of  claim 48  in which the lats protein is a human lats protein.  
     
     
         50 . A pharmaceutical composition comprising a therapeutically effective amount of the fragment of  claim 12;  and a pharmaceutically acceptable carrier.  
     
     
         51 . A pharmaceutical composition comprising a therapeutically effective amount of the protein of  claim 14;  and a pharmaceutically acceptable carrier.  
     
     
         52 . A pharmaceutical composition comprising a therapeutically effective amount of the chimeric protein of  claim 17;  and a pharmaceutically acceptable carrier.  
     
     
         53 . A pharmaceutical composition comprising a therapeutically effective amount of the nucleic acid of  claim 23;  and a pharmaceutically acceptable carrier.  
     
     
         54 . A pharmaceutical composition comprising a therapeutically effective amount of the nucleic acid of  claim 35;  and a pharmaceutically acceptable carrier.  
     
     
         55 . A pharmaceutical composition comprising a therapeutically effective amount of the recombinant cell of  claim 36;  and a pharmaceutically acceptable carrier.  
     
     
         56 . A pharmaceutical composition comprising a therapeutically effective amount of an antibody that immunospecifically binds to a lats protein; and a pharmaceutically acceptable carrier.  
     
     
         57 . A pharmaceutical composition comprising a therapeutically effective amount of a fragment or derivative of an antibody that immunospecifically binds to a lats protein, said fragment or derivative containing the binding domain of the antibody; and a pharmaceutically acceptable carrier.  
     
     
         58 . A method of treating or preventing a disease or disorder involving cell overproliferation in a subject comprising administering to a subject in which such treatment or prevention is desired a therapeutically effective amount of a molecule that promotes lats function.  
     
     
         59 . The method according to  claim 58  in which the disease or disorder is a malignancy.  
     
     
         60 . The method according to  claim 59  in which the disease or disorder is selected from the group consisting of bladder cancer, breast cancer, colon cancer, leukemia, lung cancer, melanoma, pancreatic cancer, sarcoma, and uterine cancer.  
     
     
         61 . The method according to  claim 58  in which the subject is a human.  
     
     
         62 . The method according to  claim 58  in which the disease or disorder is selected from the group consisting of premalignant conditions, benign tumors, hyperproliferative disorders, and benign dysproliferative disorders.  
     
     
         63 . The method according to  claim 58  in which the molecule that promotes lats function is selected from the group consisting of a lats protein, a lats derivative or analog that is active in inhibiting cell proliferation, a nucleic acid encoding a lats protein, and a nucleic acid encoding a lats derivative or analog that is active in inhibiting cell proliferation.  
     
     
         64 . The method according to  claim 58  in which the molecule that promotes lats function is a lats derivative or analog that comprises a kinase domain of a lats protein that has been mutated so as to be dominantly active.  
     
     
         65 . The method according to  claim 58  in which the molecule that promotes lats function is the protein of  claim 14 .  
     
     
         66 . A method of treating or preventing a disease or disorder involving a deficiency in cell proliferation or in which cell proliferation is desirable for treatment or prevention in a subject comprising administering to a subject in which such treatment or prevention is desired a therapeutically effective amount of a molecule that inhibits lats function.  
     
     
         67 . The method according to  claim 66  in which the molecule that inhibits lats function is selected from the group consisting of an anti-lats antibody or a fragment or derivative thereof containing the binding region thereof, a lats derivative or analog that is capable of being bound by an anti-lats antibody and that is a dominant-negative protein kinase, a lats antisense nucleic acid, and a nucleic acid comprising at least a portion of a lats gene into which a heterologous nucleotide sequence has been inserted such that said heterologous sequence inactivates the biological activity of the at least a portion of the lats gene, in which the lats gene portion flanks the heterologous sequence so as to promote homologous recombination with a genomic lats gene.  
     
     
         68 . The method according to  claim 66  in which the molecule that inhibits lats function is an oligonucleotide which (a) consists of at least six nucleotides; (b) comprises a sequence complementary to at least a portion of an RNA transcript of a lats gene; and (c) is hybridizable to the RNA transcript under moderately stringent conditions.  
     
     
         69 . The method according to  claim 66  in which the disease or disorder is selected from the group consisting of degenerative disorders, growth deficiencies, hypoproliferative disorders, physical trauma, lesions, and wounds.  
     
     
         70 . An isolated oligonucleotide consisting of at least six nucleotides, and comprising a sequence complementary to at least a portion of an RNA transcript of a lats gene, which oligonucleotide is hybridizable to the RNA transcript under moderately stringent conditions.  
     
     
         71 . A pharmaceutical composition comprising the oligonucleotide of  claim 70;  and a pharmaceutically acceptable carrier.  
     
     
         72 . A method of inhibiting the expression of a nucleic acid sequence encoding a lats protein in a cell comprising providing the cell with an effective amount of the oligonucleotide of  claim 70 .  
     
     
         73 . A method of diagnosing a disease or disorder characterized by an aberrant level of lats RNA or protein in a subject, comprising measuring the level of lats RNA or protein in a sample derived from the subject, in which an increase or decrease in the level of lats RNA or protein, relative to the level of lats RNA or protein found in an analogous sample not having the disease or disorder indicates the presence of the disease or disorder in the subject.  
     
     
         74 . A method of diagnosing or screening for the presence of or a predisposition for developing a disease or disorder involving cell overproliferation in a subject comprising measuring the level of lats protein, lats RNA or lats functional activity in a sample derived from the subject, in which a decrease in the level of lats protein, lats RNA, or lats functional activity in the sample, relative to the level of lats protein, lats RNA, or lats functional activity found in an analogous sample not having the disease or disorder or a predisposition for developing the disease or disorder, indicates the presence of the disease or disorder or a predisposition for developing the disease or disorder.  
     
     
         75 . A method of diagnosing or screening for the presence of or a predisposition for developing a disease or disorder involving cell overproliferation in a subject comprising detecting one or more mutations in lats DNA, RNA or protein derived from the subject in which the presence of said one or more mutations indicates the presence of the disease or disorder or a predisposition for developing the disease or disorder.  
     
     
         76 . A method of diagnosing or screening for the presence of or a predisposition for developing a disease or disorder involving a deficiency in cell proliferation or in which cell proliferation is desirable for treatment or prevention in a subject comprising measuring the level of lats protein, lats RNA or lats functional activity in a sample derived from the subject, in which an increase in the level of lats protein, lats RNA, or lats functional activity in the sample, relative to the level of lats protein, lats RNA, or lats functional activity found in an analogous sample not having the disease or disorder or a predisposition for developing the disease or disorder, indicates the presence of the disease or disorder or a predisposition for developing the disease or disorder.  
     
     
         77 . A kit comprising in one or more containers a molecule selected from the group consisting of an anti-lats antibody, a nucleic acid probe capable of hybridizing to a lats RNA, or a pair of nucleic acid primers capable of priming amplification of at least a portion of a lats nucleic acid.  
     
     
         78 . A kit comprising in a container a therapeutically effective amount of a lats protein.  
     
     
         79 . A method of increasing cell growth in animals or plants comprising inhibiting lats expression or activity in said animals or plants.  
     
     
         80 . The method of  claim 79  in which cell growth is increased in an edible plant.  
     
     
         81 . The method of  claim 79  in which cell growth is increased in a farm animal.  
     
     
         82 . A method of identifying a molecule that specifically binds to a ligand selected from the group consisting of a lats protein, a fragment of a lats protein comprising a domain of the protein, and a nucleic acid encoding the protein or fragment, comprising 
 (a) contacting said ligand with a plurality of molecules under conditions conducive to binding between said ligand and the molecules; and    (b) identifying a molecule within said plurality that specifically binds to said ligand.    
     
     
         83 . A recombinant non-human animal or plant that is the product of a process comprising introducing a nucleic acid encoding at least a domain of a lats protein into the plant or animal.  
     
     
         84 . A recombinant plant containing and capable of expressing a lats antisense nucleic acid.  
     
     
         85 . A recombinant non-human animal or plant in which a lats gene has been inactivated by a method comprising introducing a nucleic acid into the plant or animal or an ancestor thereof, which nucleic acid comprises a non-lats sequence flanked by lats genomic sequences that promote homologous recombination.  
     
     
         86 . A method of identifying a tumor suppressor gene comprising (a) identifying an overproliferation phenotype in a genetic mosaic; and (b) isolating a gene that is mutated in cells exhibiting said overproliferation phenotype.  
     
     
         87 . The method of  claim 86  in which the genetic mosaic is an animal containing (a) a nucleic acid encoding and capable of expressing a recombinase, and (b) intrachromosomal insertions of a target site at which the recombinase promotes recombination, on the homologous arms of both of a set of parental chromosomes; and the genetic mosaic has been produced by a method comprising inducing expression of the recombinase.  
     
     
         88 . The method of  claim 87  in which the recombinase is an FLP recombinase, and the target site is an FRT site.  
     
     
         89 . The method according to  claim 87  in which the recombinase is a Cre recombinase, and the target site is a lox site.  
     
     
         90 . The method of  claim 86  in which the overproliferation phenotype is the formation of overproliferated outgrowth tissue.  
     
     
         91 . The method of  claim 86  in which the overproliferation phenotype is the formation of a normal structure of larger than normal size.  
     
     
         92 . A non-human mammal comprising (a) a nucleic acid sequence encoding a recombinase operably linked to a promoter; and (b) intrachromosomal insertions into the homologous arms of both of a set of parental chromosomes, of a target site at which the recombinase can promote recombination.  
     
     
         93 . The mammal of  claim 92  which is heterozygous for an induced mutation.  
     
     
         94 . The mammal of  claim 93  in which the sequence encoding the recombinase is operably linked to an inducible promoter.  
     
     
         95 . A method of making a genetic mosaic comprising inducing expression of the recombinase in the mammal of  claim 93 .  
     
     
         96 . A method for identifying a gene with an identifiable mutant phenotype comprising: 
 (a) identifying a mutant phenotype in a genetic mosaic animal, said genetic mosaic animal having been produced by a method comprising recombinantly expressing a recombinase within a cell of the animal so as to promote recombination at intrachromosomally inserted target sites on the homologous arms of both of a set of parental chromosomes; and    (b) isolating a gene that is mutated in cells exhibiting said mutant phenotype.    
     
     
         97 . A method for identifying a gene with an identifiable mutant phenotype comprising: 
 (a) identifying a mutant phenotype in a cultured cell, said cultured cell having been produced by a method comprising recombinantly expressing a recombinase within said cultured cell so as to promote recombination at intrachromosomally inserted target sites on the homologous arms of both of a set of parental chromosomes; and    (b) isolating a gene that is mutated in cells exhibiting said mutant phenotype.    
     
     
         98 . The method of  claim 97  in which the mutant phenotype is a transformed phenotype.  
     
     
         99 . The mammal of  claim 92  in which the promoter is not a native recombinase gene promoter.  
     
     
         100 . A method of inhibiting cellular senescence in a subject comprising administering to a subject in which such inhibition is desired an amount of a molecule that inhibits lats function, effective to inhibit cellular senescence.  
     
     
         101 . A method of inhibiting cellular senescence in cells in vitro comprising contacting cells in vitro with an amount of a molecule that inhibits lats function, effective to inhibit cellular senescence.  
     
     
         102 . The method according to  claim 100  in which the molecule that inhibits lats function is selected from the group consisting of an anti-lats antibody or a fragment or derivative thereof containing the binding region thereof, a lats derivative or analog that is capable of being bound by an anti-lats antibody and that is a dominant-negative protein kinase, a lats antisense nucleic acid, and a nucleic acid comprising at least a portion of a lats gene into which a heterologous nucleotide sequence has been inserted such that said heterologous sequence inactivates the biological activity of the at least a portion of the lats gene, in which the lats gene portion flanks the heterologous sequence so as to promote homologous recombination with a genomic lats gene.  
     
     
         103 . The method according to  claim 101  in which the molecule that inhibits lats function is selected from the group consisting of an anti-lats antibody or a fragment or derivative thereof containing the binding region thereof, a lats derivative or analog that is capable of being bound by an anti-lats antibody and that is a dominant-negative protein kinase, a lats antisense nucleic acid, and a nucleic acid comprising at least a portion of a lats gene into which a heterologous nucleotide sequence has been inserted such that said heterologous sequence inactivates the biological activity of the at least a portion of the lats gene, in which the lats gene portion flanks the heterologous sequence so as to promote homologous recombination with a genomic lats gene.