Claims
- 1. An isolated nucleic acid molecule, comprising a nucleotide sequence encoding an IκB kinase (IKK) subunit, IKKβ, which phosphorylates the inhibitor of NF-κB (IκBα) on serine-32 and serine-36 and has an apparent molecular mass of about 87 kiloDaltons; or a nucleotide sequence complementary thereto.
- 2. The nucleic acid molecule of claim 1, comprising the nucleotide sequence shown in SEQ ID NO: 14.
- 3. The nucleic acid molecule of claim 1, comprising a nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO: 15.
- 4. A vector, comprising the nucleic acid molecule of claim 1.
- 5. The vector of claim 4, which is an expression vector.
- 6. The vector of claim 4, which is a viral vector.
- 7. A host cell containing the vector of claim 4.
- 8. A polynucleotide, comprising a nucleotide sequence selected from the group consisting of:
a) a nucleotide sequence encoding at least three contiguous amino acids of SEQ ID NO: 15; b) a nucleotide sequence complementary to a nucleotide sequence encoding at least three contiguous amino acids of SEQ ID NO: 15; and c) a mixture of nucleotide sequence, comprising a nucleotide sequence encoding at least three contiguous amino acids of SEQ ID NO: 15, and a nucleotide sequence complementary thereto, wherein said mixture comprises single stranded polynucleotides or double stranded polynucleotides.
- 9. An antisense nucleic acid molecule, comprising a polynucleotide sequence complementary to at least nine contiguous nucleotides of SEQ ID NO: 14 or complementary to a nucleotide sequence encoding at least three contiguous amino acids of SEQ ID NO: 15.
- 10. An isolated IκB kinase (IKK) subunit, IKKβ, wherein said IKKβ phosphorylates serine-32 and serine-36 of IκBα and has an apparent molecular mass of 87 kiloDaltons as determined by SDS-polyacrylamide gel electrophoresis in an 8% gel under reducing conditions.
- 11. The isolated IKKβ of claim 10, comprising an amino acid sequence as shown in SEQ ID NO: 15.
- 12. A peptide, comprising at least three contiguous amino acids of SEQ ID NO: 15.
- 13. An antibody that specifically binds to an epitope comprising an amino acid of SEQ ID NO: 15, provided said epitope is not present in SEQ ID NO: 2.
- 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 IκB 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 IκB protein.
- 23. The method of claim 22, wherein said IκB protein is selected from the group consisting of IκBα and IκBβ.
- 24. The method of claim 16, wherein said second protein is a subunit of a 300 kDa IκB kinase complex or a 900 kDa IκB 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 IκB protein selected from the group consisting of a mutant IκBα containing amino acid substitutions for serine-32 and for serine-36 and a mutant IκBβ containing amino acid substitutions for serine-19 and for serine-23.
- 29. A method for identifying an agent that alters IκB 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 IκB kinase complex or a 900 kDa IκB kinase complex.
- 33. The method of claim 32, wherein said composition comprises IKKα or IKKβ.
- 34. A method of obtaining isolated IκB kinase (IKK) from a sample containing the IKK, comprising the steps of:
a) contacting the sample containing the IKK with an antibody the specifically binds an epitope of the IKK; 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 33, wherein said catalytic subunit is IKKα or IKKβ.
- 37. The method of claim 34, wherein said antibody specifically binds a 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-κB activity in a cell, comprising contacting the cell with an agent that alters the association of an IκB kinase (IKK) or an IKK catalytic subunit and a second protein.
- 41. A method of modulating NF-κB activity in a cell, comprising contacting the cell with an agent that alters the activity of an IκB kinase.
- 42. A method of modulating NF-κB activity in a cell, 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.
Parent Case Info
[0001] This application is based on, and claims the benefit of, U.S. Provisional Application No. 60/061,470, filed Oct. 9, 1997, the entire contents of which is herein incorporated by reference.
Government Interests
[0002] This invention was made with government support under grant number CA50528 awarded by the National Institutes of Health. The government has certain rights in the invention.
Provisional Applications (1)
|
Number |
Date |
Country |
|
60061470 |
Oct 1997 |
US |
Divisions (1)
|
Number |
Date |
Country |
Parent |
09168629 |
Oct 1998 |
US |
Child |
09796872 |
Feb 2001 |
US |