Kinase capable of site-specific phosphorylation of ikba

Abstract

The present invention relates, in general, to a kinase which in its activated state is capable of site-specific phosphorylation of IκBα, IκBα kinase. In particular, the present invention relates to the purified kinase, purified polypeptide subunits of the kinase, nucleic acid molecules coding for the purified polypeptide subunits; recombinant nucleic acid molecules; cells containing the recombinant nucleic acid molecules; antibodies having binding affinity specifically to the kinase or its polypeptide subunits; hybridomas containing the antibodies; nucleic acid probes for the detection of the nucleic acid encoding the kinase; a method of detecting nucleic acids encoding the kinase or polypeptides of the kinase in a sample; and kits containing nucleic acid probes or antibodies. This invention further relates to bioassays using the nucleic acid sequence, protein or antibodies of this invention to diagnose, assess, or prognose a mammal afflicted with an undesired activation of NF-κB. This invention also relates to ligands, agonists, and antagonists of the kinase, and diagnostic and therapeutic uses thereof. This invention also relates to bioassays using the kinase or polypeptides of the kinase of this invention to identify ligands, agonists, and antagonists. More specifically, this invention relates to selective inhibitors of the kinase and to structure-based design of ligands, agonists, and antagonists of the kinase. This invention further relates to gene therapy using the nucleic acids of the invention.

What is claimed is:  
     
         1 . A purified kinase which phosphorylates IκBα at serine residues 32 and 36.  
     
     
         2 . A purified kinase which, when activated, phosphorylates IκBα at serine residues 32, and 36.  
     
     
         3 . The kinase of  claim 2  which, when ubiquitinated, phosphorylates IκBα at serine residues 32 and 36.  
     
     
         4 . The kinase of  claim 2  which, when phosphorylated, phosphorylates IκBα at serine residues 32 and 36.  
     
     
         5 . The kinase of any one of claims  1  or  2  wherein said kinase comprises an amino acid sequence which is at least 60% homologous to the amino acid sequence of any one of FIGS.  21 A-D.  
     
     
         6 . The kinase of  claim 5  comprising an amino acid sequence which is at least 75% homologous to the amino acid sequence of any one of FIGS.  21 A-D.  
     
     
         7 . The kinase of  claim 5  comprising an amino acid sequence which is at least 90% homologous to the amino acid sequence of any one of FIGS.  21 A-D.  
     
     
         8 . The kinase of  claim 5  comprising an amino acid sequence comprising the amino acid sequence of any one of FIGS.  21 A-D.  
     
     
         9 . The kinase of any one of claims  1  or  2  wherein said kinase is encoded by a nucleic acid molecule which hybridizes to a second nucleic acid molecule having the nucleotide sequence of any one of FIGS.  22 A-B.  
     
     
         10 . The kinase of  claim 9  wherein said kinase is encoded by a nucleic acid molecule comprising the nucleotide sequence of any one of FIGS.  22 A-B.  
     
     
         11 . A purified polypeptide subunit of the kinase according to  claim 2 .  
     
     
         12 . A purified polypeptide subunit of the kinase according to  claim 11  wherein said subunit, when active, phosphorylates IκBα.  
     
     
         13 . A purified polypeptide subunit of the kinase according to  claim 1  which phosphorylates IκBα.  
     
     
         14 . A purified polypeptide subunit of the kinase according to  claim 11  wherein said subunit is selected from the group consisting of p31, p33, p36, p38, p40, p43, p50, p55, p62, p70 and p85.  
     
     
         15 . An isolated nucleic acid molecule encoding the kinase according to  claim 2 .  
     
     
         16 . An isolated nucleic acid molecule encoding the polypeptide subunit according to any one of claims  11  or  12 .  
     
     
         17 . An isolated nucleic acid molecule which hybridizes to a second nucleic acid molecule having the nucleotide sequence set forth in any one of FIGS.  22 A-B.  
     
     
         18 . An isolated nucleic acid molecule which comprises the nucleotide sequence set forth in any one of FIGS.  22 A-B.  
     
     
         19 . An isolated nucleic acid molecule according to any one of claims  15  or  16  which hybridizes to a second nucleic acid molecule having the nucleotide sequence set forth in any one of FIGS.  22 A-B.  
     
     
         20 . An isolated nucleic acid molecule according to any one of claims  15  or  16  which comprises the nucleotide sequence set forth in any one of FIGS.  22 A-B.  
     
     
         21 . An isolated nucleic acid molecule consisting of 10 to 50 nucleotides which hybridizes preferentially to RNA or DNA of a kinase which when activated phosphorylates IκBα at serine residues 32 and 36, wherein said nucleic acid molecule is or is complementary to a nucleotide sequence consisting of at least 10 consecutive nucleotides from the nucleic acid molecule according to  claim 16 .  
     
     
         22 . A method of detecting nucleic acid encoding a kinase or subunit thereof which phosphorylates IκBα at serine residues 32 and 36 in a sample comprising: 
 (a) contacting said sample with a nucleic acid molecule according to any one of claims  15 - 21  under conditions such that hybridization occurs, and  
 (b) detecting the presence of said molecule bound to nucleic acid encoding said kinase.  
 
     
     
         23 . A kit for detecting the presence of nucleic acid encoding a kinase or subunit thereof which phosphorylates IκBα at serine residues 32 and 36 in a sample comprising at least one container means having disposed therein the nucleic acid molecule according to any one of claims  15 - 21 .  
     
     
         24 . A recombinant nucleic acid molecule comprising 5′ to 3′, a promoter effective to initiate transcription in a host cell and the isolated nucleic acid molecule according to any one of claims  15 ,  16 ,  17 , or  18 .  
     
     
         25 . A recombinant nucleic acid molecule comprising a vector and the isolated nucleic acid molecule according to any one of claims  15 ,  16 ,  17 , or  18 .  
     
     
         26 . A host cell that contains the recombinant nucleic acid molecule according to any one of claims  24  or  25 .  
     
     
         27 . A non-human organism that contains the recombinant nucleic acid molecule according to any one of claims  24  or  25 .  
     
     
         28 . An antibody having binding affinity specifically to the kinase or subunit thereof according to any one of claims  1 ,  2 ,  11 ,  12  or  13 .  
     
     
         29 . A method of detecting a kinase or subunit thereof in a sample, comprising: 
 (a) contacting said antibody according to  claim 28 , under conditions such that immunocomplexes form, and    (b) detecting the presence of said antibody bound to said kinase or subunit thereof.    
     
     
         30 . A diagnostic kit comprising: 
 (a) a first container means containing the antibody according to claim  28 ; and    (b) second container means containing a conjugate comprising a binding partner of said antibody and a label.    
     
     
         31 . A hybridoma which produces the antibody according to  claim 30 .  
     
     
         32 . A bioassay for assessing candidate drugs or ligands of the kinase according to  claim 1  or the subunit according to  claim 13  comprising: 
 (a) contacting a candidate drug or ligand with a sample containing the kinase according to  claim 1  or kinase subunit according to claim  13 ; and  
 (b) evaluating the biological activity modified by said contact  
 wherein a reduction in the amount of biological activity in the presence of the candidate drug or ligand indicates that the candidate drug or ligand is an inhibitor of kinase activity.  
 
     
     
         33 . The bioassay according to  claim 32 , wherein said biological activity comprises the amount of phosphorylated substrate that is produced.  
     
     
         34 . The bioassay according to  claim 33 , wherein the substrate is labeled IκBα.  
     
     
         35 . A ligand capable of selective binding to the kinase or subunit thereof according to any one of claims  1 ,  2 ,  11 ,  12  or  13 .  
     
     
         36 . The ligand of  claim 35  which is a selective inhibitor or antagonist of kinase activity.  
     
     
         37 . The inhibitor of  claim 36  which is obtained by the bioassay of  claim 32 .  
     
     
         38 . A method of treatment of a disease in a mammal requiring modulation of NF-KB, comprising administering a therapeutically effective amount of purified ligand of  claim 35 ,  36  or  37  to a mammal afflicted with said disease.  
     
     
         39 . A method of treatment of a disease in a mammal wherein NF-KB activity is low as compared to activity in a normal mammal, comprising administering a therapeutically effective amount of the nucleic acid according to any one of claims  15  or  16  to a mammal afflicted with said disease.  
     
     
         40 . The kinase according to any one of claims  1  or  2 , wherein said kinase is purified by chromatographic purification of cell extracts.  
     
     
         41 . The kinase according to claim  40 , wherein said extracts are cell cytoplasmic extracts.  
     
     
         42 . The kinase according to claim  40 , wherein said chromatographic purification comprises ion-exchange chromatography and size exclusion chromatography.