IKK-alpha proteins, nucleic acids and methods

Abstract

The invention provides methods and compositions relating to an IκB kinase, IKK-α, and related nucleic acids. The polypeptides may be produced recombinantly from transformed host cells from the disclosed IKK-α encoding nucleic acids or purified from human cells. The invention provides isolated IKK-α hybridization probes and primers capable of specifically hybridizing with the disclosed IKK-α genes, IKK-α-specific binding agents such as specific antibodies, and methods of making and using the subject compositions in diagnosis, therapy and in the biopharmaceutical industry.

1 . An isolated IKK-αGlu 543  polypeptide, comprising at least 10 consecutive amino acid residues of the amino acid sequence set forth as SEQ ID NO:4, which consecutive amino acid residues comprise the amino acid residue 543 (Glu) of SEQ ID NO:4.  
     
     
         2 . An isolated polypeptide according to  claim 1 , wherein said polypeptide has an activity selected from at least one of: a kinase or kinase inhibitory activity, a NIK-binding or binding inhibitory activity, an IκB-binding or binding inhibitory activity and an NFκB activating or inhibitory activity.  
     
     
         3 . An isolated or recombinant IKK-αA 1628  nucleic acid comprising at least 24 consecutive nucleotides of the nucleotide sequence set forth as SEQ ID NO:3, which consecutive polynucleotides comprise the polynucleotide 1628 (A) of SEQ ID NO:3.  
     
     
         4 . A recombinant nucleic acid encoding a polypeptide according to  claim 1 .  
     
     
         5 . A cell comprising a nucleic acid according to  claim 4 .  
     
     
         6 . A method of making an isolated polypeptide according to  claim 1 , said method comprising steps: introducing a nucleic acid according to  claim 4  into a host cell or cellular extract, incubating said host cell or extract under conditions whereby said nucleic acid is expressed as a transcript and said transcript is expressed as a translation product comprising said polypeptide, and isolating said translation product.  
     
     
         7 . A method of screening for an agent which modulates the interaction of an IKK polypeptide to a binding target, said method comprising the steps of: 
 incubating a mixture comprising: 
 an isolated polypeptide according to  claim 1 , a binding target of said polypeptide, and a candidate agent;  
   under conditions whereby, but for the presence of said agent, said polypeptide specifically binds said binding target at a reference affinity;    detecting the binding affinity of said polypeptide to said binding target to determine an agent-biased affinity, wherein a difference between the agent-biased affinity and the reference affinity indicates that said agent modulates the binding of said polypeptide to said binding target.    
     
     
         8 . A method according to  claim 7 , wherein said binding target is a natural intracellular substrate and said reference and agent-biased binding affinity is detected as phosphorylation of said substrate.  
     
     
         9 . A method of screening for an agent which modulates the interaction of an IKK polypeptide to a binding target, said method comprising the steps of: 
 incubating a mixture comprising: an isolated polypeptide according to  claim 1  comprising SEQ ID NO: 2 or 4, or a deletion mutant thereof retaining IκB kinase activity, an IκB polypeptide comprising at least a six residue domain of a natural IκB comprising at least one of Ser32 and Ser 36, and a candidate agent;    under conditions whereby, but for the presence of said agent, said polypeptide specifically phosphorylates said IκB polypeptide at at least one of said Ser32 and Ser36 at a reference activity;    detecting the polypeptide-induced phosphorylation of said IκB polypeptide at at least one of said Ser32 and Ser36 to determine an agent-biased activity, wherein a difference between the agent-biased activity and the reference activity indicates that said agent modulates the ability of said polypeptide to specifically phosphorylate a IκB polypeptide.    
     
     
         10 . A method for modulating signal transduction involving IκB in a cell, said method comprising the step of modulating IKK-α (SEQ ID NO:4) kinase activity.  
     
     
         11 . The method of  claim 10 , wherein said modulating step comprises contacting the cell with a serine/threonine kinase inhibitor.  
     
     
         12 . An antibody which specifically binds a polypeptide according to  claim 1.