Elongation factor-2 kinase (EF-2 kinase) and methods of use therefor

Abstract

A new superfamily of protein kinases has been discovered that centers around eukaryotic elongation factor-2 kinase (eEF-2 kinase). The protein kinases of this new superfamily have the following characteristics: 1) sequence similarity to eEF-2 kinase; 2) no sequence similarity to the protein kinases of either the serine/threonine/tyrosine kinase or histidine kinase superfamily; and, 3) specifically phosphorylates α-helical regions of proteins as opposed to β-turns, as seen in all other protein kinases. Assays have been developed utilizing eEF-2 kinase and a phosphorylation target consisting of a novel α-helical 16-amino acid peptide sequence to facilitate high-throughput screening for compounds that can specifically inhibit this protein kinase that has been implicated tumor growth and other hyperproliferitive disorders. Additionally, the disclosed invention includes assessing eEF-2 kinase levels for diagnostic purposes, and therapeutic formulations to inhibit eEF-2 kinase activity.

What is claimed is:  
     
         1 . A protein kinase which is a member of a superfamily, said protein kinase being characterized by: 
 A. greater than 40% sequence similarity with eEF-2 kinase from any organism; and.    B. phosphorylates an amino acid within an alpha helical domain of its target protein.    
     
     
         2 . A protein kinase of  claim 1  which phosphorylates eukaryotic elongation factor-2 (eEF-2), and is designated as eukaryotic elongation factor-2 kinase (eEF-2 kinase).  
     
     
         3 . A protein kinase of  claim 1  which phosphorylates eukaryotic myosin heavy chain (MHC), and is designated as myosin heavy chain kinase (MHCK).  
     
     
         4 . A protein kinase of  claim 1  that phosphorylates a peptide sequence derived from the phosphorylation site of a target protein.  
     
     
         5 . A peptide sequence having SEQ ID NO: 20.  
     
     
         6 . A protein kinase of  claim 1  which is a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 4, and SEQ ID NO: 10, and fragments thereof.  
     
     
         7 . A protein kinase of  claim 1  which is derived from mammalian cells.  
     
     
         8 . A protein kinase of  claim 1  labeled with a detectable label.  
     
     
         9 . A protein kinase of  claim 8  wherein the label is selected from enzymes, chemicals which fluoresce, and radioactive elements.  
     
     
         10 . An antibody to the protein kinase of  claim 1 .  
     
     
         11 . An antibody to the phosphorylated form of the target protein of  claim 1 .  
     
     
         12 . An antibody to the phosphorylated form of the peptide of  claim 5 .  
     
     
         13 . The antibody of  claim 10 ,  11  or  12  which is a polyclonal antibody.  
     
     
         14 . The antibody of  claim 10 ,  11  or  12  which is a monoclonal antibody.  
     
     
         15 . An immortal cell line that produces a monoclonal antibody according to  claim 14 .  
     
     
         16 . The antibody of  claim 10 ,  11  or  12  labeled with a detectable label.  
     
     
         17 . The antibody of  claim 10 ,  11  or  12  wherein the label is selected from enzymes, chemicals which fluoresce and radioactive elements.  
     
     
         18 . A DNA sequence which encodes eEF-2 kinase, or a fragment thereof, selected from the group consisting of: 
 (A) the DNA sequences of FIG. 5 (SEQ ID NO: 1);    (B) the DNA sequences of FIG. 5 (SEQ ID NO: 3);    (C) the DNA sequences of FIG. 5 (SEQ ID NO: 9);    (D) DNA sequences that hybridize to any of the foregoing DNA sequences under standard hybridization conditions;    (E) DNA sequences that code for expression of an amino acid sequence encoded by any of the foregoing DNA sequences.    (F) degenerate variants thereof;    (G) alleles thereof; and,    (H) hybridizable fragments thereof.    
     
     
         19 . A recombinant DNA molecule comprising a DNA sequence which encodes eEF-2 kinase, or a fragment thereof, selected from the group consisting of: 
 (A) the DNA sequences of FIG. 5 (SEQ ID NO: 1);    (B) the DNA sequences of FIG. 5 (SEQ ID NO: 3);    (C) the DNA sequences of FIG. 5 (SEQ ID NO: 9);    (D) DNA sequences that hybridize to any of the foregoing DNA sequences under standard hybridization conditions;    (E) DNA sequences that code for expression of an amino acid sequence encoded by any of the foregoing DNA sequences.    (F) degenerate variants thereof;    (G) alleles thereof; and,    (H) hybridizable fragments thereof.    
     
     
         20 . The recombinant DNA molecule of either of claims  18  or  19 , wherein said DNA sequence is operatively linked to an expression control sequence.  
     
     
         21 . The recombinant DNA molecule of  claim 20 , wherein said expression control sequence is selected from the group consisting of the early or late promoters of SV40 or adenovirus, the lac system, the trp system, the TAC system, the TRC system, the major operator and promoter regions of phage λ, the control regions of fd coat protein, the promoter for 3-phosphoglycerate kinase, the promoters of acid phosphatase and the promoters of the yeast α-mating factors.  
     
     
         22 . A probe capable of screening for eEF-2 kinase in alternate species prepared from the DNA sequence of  claim 18 .  
     
     
         23 . A probe capable of screening for members of the protein kinase superfamily of  claim 1  prepared from the DNA sequence of  claim 18 .  
     
     
         24 . A unicellular host transformed with a recombinant DNA molecule comprising a DNA sequence or degenerate variant thereof, which encodes a protein kinase, or a fragment thereof, selected from the group consisting of: 
 (A) the DNA sequences of FIG. 5 (SEQ ID NO: 1);    (B) the DNA sequences of FIG. 5 (SEQ ID NO: 3);    (C) the DNA sequences of FIG. 5 (SEQ ID NO: 9);    (D) DNA sequences that hybridize to any of the foregoing DNA sequences under standard hybridization conditions; and    (E) DNA sequences that code on expression for an amino acid sequence encoded by any of the foregoing DNA sequences;    wherein said DNA sequence is operatively linked to an expression control sequence.    
     
     
         25 . The unicellular host of  claim 24  wherein the unicellular host is selected from the group consisting of  E. coli,  Pseudomonas, Bacillus, Streptomyces, yeasts, CHO, R1.1, B-W, L-M, COS 1, COS 7, BSC1, BSC40, and BMT10 cells, plant cells, insect cells, and human cells in tissue culture.  
     
     
         26 . A method for detecting eEF-2 kinase and assessing eEF-2 kinase levels by: 
 A. contacting a biological sample from a mammal in which the presence or activity of said eEF-2 kinase is suspected with a binding partner of said eEF-2 kinase under conditions that allow binding of said eEF-2 kinase to said binding partner to occur; and,    B. detecting whether binding has occurred, and to what degree, between said eEF-2 kinase from said sample and the binding partner;    wherein the detection of binding indicates that presence or activity of said eEF-2 kinase in said sample, and indicates a level of said eEF-2 kinase in the sample.    
     
     
         27 . An assay system for screening drugs and other agents for ability to modulate eEF-2 kinase activity. comprising a predetermined amount of eEF-2 kinase mixed with varying amounts of drug or agent, along with target protein and ATP; wherein detection is via either a detectable label on the γ-phosphate of ATP, or on an antibody directed against the phosphorylated target protein.  
     
     
         28 . The assay system of  claim 27  wherein the label on the γ-phosphate of ATP comprises one of the following: 
 A.  32 P  
 B.  33 P  
 C.  35 S  
 D. a biotinylated phosphate moiety; or,  
 E. a fluorescent phosphate moiety.  
 
     
     
         29 . The assay system of  claim 27  wherein the label on the antibody comprises one of the following: 
 A. an enzyme detectable with calorimetric, fluorescent, or chemiluminescent substrates, such as alkaline phosphatase or horseradish peroxidase;  
 B. a biotin moiety;  
 C. a fluorescent moiety; or,  
 D. a radioactive moiety chosen from the following group of isotopes:  3 H,  14 C,  32 P,  33 P,  35 S,  36 Cl,  51 Cr,  57 Co,  58 Co,  59 Fe,  90 Y,  125 I,  131 I, and  186 Re.  
 
     
     
         30 . An assay system for screening drugs and other agents for ability to modulate eEF-2 kinase activity, comprising: 
 A. culturing an observable cellular test colony inoculated with a drug or agent;    B. harvesting a supernatant from said cellular test colony; and,    C. examining said supernatant for the presence of said eEF-2 kinase activity wherein an increase or a decrease in a level of said eEF-2 kinase activity indicates the ability of a drug to modulate the activity of said eEF-2 kinase.    
     
     
         31 . A test kit for assessing the level of eEF-2 kinase activity in a eukaryotic cellular sample, comprising: 
 A. a predetermined amount of a detectably labelled specific binding partner of eEF-2 kinase.    B. other reagents; and,    C. directions for use of said kit.    
     
     
         31 . The test kit of  claim 31  wherein said labeled immunochemically reactive component is selected from the group consisting of polyclonal antibodies to eEF-2 kinase, monoclonal antibodies to eEF-2 kinase, fragments thereof, and mixtures thereof.  
     
     
         32 . A method of preventing and/or treating cellular debilitations, derangements and/or dysfunctions and/or other disease states in mammals, comprising administering to a mammal a therapeutically effective amount of a material selected from the following group: 
 A. peptides that inhibit eEF-2 kinase;    B. antibodies against eEF-2 kinase; and,    C. other drugs or agents that specifically inhibit eEF-2 kinase.    
     
     
         33 . A pharmaceutical composition for the treatment of cellular debilitation, derangement and/or dysfunction in mammals, comprising: 
 A. a therapeutically effective amount of a material selected from the group consisting of: peptides that inhibit eEF-2 kinase; antibodies against eEF-2 kinase; and, other drugs or agents that specifically inhibit eEF-2 kinase; and,    B. a pharmaceutically acceptable carrier.    
     
     
         34 . A recombinant virus transformed with the DNA molecule, or a derivative or fragment thereof, in accordance with  claim 18 .  
     
     
         35 . A recombinant virus transformed with the DNA molecule, or a derivative or fragment thereof, in accordance with  claim 19 .  
     
     
         36 . The recombinant DNA molecule of  claim 20  comprising plasmid pGEX-3X, clone E3 or plasmid pGEX-3X, clone E4.  
     
     
         37 . An antisense nucleic acid against eEF-2 kinase mRNA comprising a nucleic acid sequence hybridizing to said mRNA.  
     
     
         38 . The antisense nucleic acid of  claim 37  which is RNA.  
     
     
         39 . The antisense nucleic acid of  claim 37  which is DNA.  
     
     
         40 . The antisense nucleic acid of  claim 37  which binds to the initiation codon of any of said mRNAs.  
     
     
         41 . A recombinant DNA molecule having a DNA sequence which, on transcription, produces an antisense ribonucleic acid against eEF-2 kinase mRNA, said antisense ribonucleic acid comprising an nucleic acid sequence capable of hybridizing to said mRNA.  
     
     
         42 . A eEF-2 kinase-producing cell line transfected with the recombinant DNA molecule of  claim 41 .  
     
     
         43 . A method for creating a cell line which exhibits reduced expression of eEF-kinase, comprising transfecting a eEF-2 kinase-producing cell line with a recombinant DNA molecule of  claim 41 .  
     
     
         44 . A ribozyme that cleaves eEF-2 kinase mRNA.  
     
     
         45 . The ribozyme of  claim 44  which is a Tetrahymena-type ribozyme.  
     
     
         46 . The ribozyme of  claim 44  which is a Hammerhead-type ribozyme.  
     
     
         47 . A recombinant DNA molecule having a DNA sequence which, upon transcription, produces the ribozyme of  claim 44 .  
     
     
         48 . A eEF-2 kinase-producing cell line transfected with the recombinant DNA molecule of  claim 47 .  
     
     
         49 . A method for creating a cell line which exhibits reduced expression of eEF-2 kinase, comprising transfecting a eEF-2 kinase-producing cell line with the recombinant DNA molecule of claim  44 .