IKB kinase, subunits thereof, and methods of using same

Abstract

The present invention provides an isolated nucleic acid molecules encoding IκB kinase (IKK) catalytic subunit polypeptides, which are associated with an IKK serine protein kinase that phosphorylates a protein (IκB) that inhibits the activity of the NF-κB transcription factor, vectors comprising such nucleic acid molecules and host cells containing such vectors. In addition, the invention provides nucleotide sequences that can bind to a nucleic acid molecule of the invention, such nucleotide sequences being useful as probes or as antisense molecules. The invention also provides isolated IKK catalytic subunits, which can phosphorylate an IκB protein, and peptide portions of such IKK subunit. In addition, the invention provides anti-IKK antibodies, which specifically bind to an IKK complex or an IKK catalytic subunit, and IKK-binding fragments of such antibodies. The invention further provides methods of substantially purifying an IKK complex, methods of identifying an agent that can alter the association of an IKK complex or an IKK catalytic subunit with a second protein, and methods of identifying proteins that can interact with an IKK complex or an IKK catalytic subunit.

1 - 12 . (canceled) 
     
     
         13 . An antibody that specifically binds to the protein set forth as SEQ ID NO: 15. 
     
     
         14 . A cell line producing the antibody of  claim 13 . 
     
     
         15 . The cell line of  claim 14 , which is a hybridoma cell line. 
     
     
         16 . A method of identifying an agent that modulates the specific association of a polypeptide of an IKB kinase (IKK) subunit and a second protein, comprising the steps of:
 a) contacting the IKK subunit and the second protein, under conditions suitable for the specific association of said IKK subunit and said second protein, with an agent suspected of being able to modulate said specific association; and   b) detecting an altered association of said IKK subunit and said second protein in the presence of said agent, wherein said altered association identifies an agent that modulates the specific association of said IKK subunit and said second protein.   
     
     
         17 . The method of  claim 16 , wherein said contacting is in an in vitro reaction and said IKK subunit is isolated. 
     
     
         18 . The method of  claim 17 , wherein said contacting is in a cell in culture. 
     
     
         19 . The method of  claim 18 , wherein said cell is selected from the group consisting of a mammalian cell and a yeast cell. 
     
     
         20 . The method of  claim 18 , wherein said altered association is detected by measuring the transcriptional activity of a reporter gene. 
     
     
         21 . The method of  claim 16 , wherein said IKK subunit is IKKα or IKKβ. 
     
     
         22 . The method of  claim 16 , wherein said second protein is an IKB protein. 
     
     
         23 . The method of  claim 22 , wherein said IKB protein is selected from the group consisting of IKBα and IKβ. 
     
     
         24 . The method of  claim 16 , wherein said second protein is a subunit of a 300 kDa IKB kinase complex or a 900 kDa IKB kinase complex. 
     
     
         25 . The method of  claim 25 , wherein said subunit is IKKα or IKKβ. 
     
     
         26 . The method of  claim 16 , wherein said agent is an organic molecule. 
     
     
         27 . The method of  claim 16 , wherein said agent is a peptide. 
     
     
         28 . The method of  claim 27 , wherein said peptide is a mutant IKB protein selected from the group consisting of a mutant IKBα containing amino acid substitutions for serine-32 and for serine-36 and a mutant IKBβ containing amino acid substitutions for serine-19 and for serine-23. 
     
     
         29 . A method for identifying an agent that alters IKB kinase (IKK) activity, comprising the steps of:
 a) incubating an isolated composition having IKK activity with an agent suspected of being able to alter said IKK activity; and   b) determining altered IKK activity of said composition in the presence of said agent, wherein said altered IKK activity identifies an agent that alters said IKK activity of said composition.   
     
     
         30 . The method of  claim 29 , wherein said agent is a protein kinase inhibitor. 
     
     
         31 . The method of  claim 29 , wherein said composition comprises an IKK subunit. 
     
     
         32 . The method of  claim 29 , wherein said composition comprises a 300 kDa IKB kinase complex or a 900 kDa IKB kinase complex. 
     
     
         33 . The method of  claim 32 , wherein said composition comprises IKKα or IKKβ. 
     
     
         34 . A method of obtaining isolated IKB kinase (IKK) from a sample containing the IKK, comprising the steps of:
 a) contacting the sample containing the IKK with an antibody that specifically binds to said IKK or binds to a tag linked thereto; and   b) obtaining isolated IKK bound to said antibody.   
     
     
         35 . The method of  claim 34 , wherein said antibody specifically binds an IKK catalytic subunit. 
     
     
         36 . The method of  claim 35 , wherein said catalytic subunit is IKKα or IKKβ. 
     
     
         37 . The method of  claim 34 , wherein said antibody specifically binds said tag linked to an IKK catalytic subunit. 
     
     
         38 . The method of  claim 37 , wherein said tag comprises a peptide tag selected from an HA tag, a HIS6 tag and a FLAG tag. 
     
     
         39 . The method of  claim 34 , wherein said antibody specifically binds an IKK complex. 
     
     
         40 . A method of modulating NF-KB activity in a cell, comprising contacting the cell with an agent that i) either alters the association of an IKB kinase (IKK) or an IKK catalytic subunit and a second protein; ii) alters the activity of said IKB kinase; or iii) alters expression of said IKK catalytic subunit. 
     
     
         41 . (canceled) 
     
     
         42 . The method of  claim 40 , comprising introducing into the cell an antisense IκB kinase subunit nucleic acid molecule. 
     
     
         43 . The method of  claim 42 , wherein said antisense IκB kinase subunit nucleic acid molecule is expressed in the cell from a vector.