Gamma subunit of cytokine responsive lkB-alpha kinase complex
a kinase complex and cytokine technology, applied in the field of molecular biology and biochemistry, can solve the problems of unsatisfactory treatment options for chronic inflammatory diseases such as asthma, rheumatoid arthritis and inflammatory bowel disease, and the side effects of prolonged treatment are associated with prolonged treatment, so as to achieve the effect of modulating nf-b activity and modulating nf-b activity
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example i
Isolation and Characterization of IKKγ
[0110] This example describes the isolation and characterization of a novel subunit of the IKK complex.
A. Purification of IKK-γ
[0111] To purify IKK to homogeneity, monoclonal antibodies specific for IKK-α were prepared. Using anti-IKK-o as an immunoaffinity reagent, the IKK complex was shown to contain nearly equal amounts of IKK-α and IKK-β in both HeLa and Jurkat cells (FIG. 1). Furthermore, passing partially purified IKK fractions through the anti-IKK-α column resulted in quantitative recovery of IKK-β (FIG. 1a), with which the antibody does not crossreact.
[0112] Large scale purification by combining the protocol described in DiDonato et al., supra, 1997, with immunoaffinity chromatography on immobilized anti-IKK-α revealed that, in addition to IKK-α and IKK-β, purified IKK contained two polypeptides of 50 and 52 kDa in size (FIG. 1b). Similar results were obtained by immunoprecipitation of 35S-labeled cell lysates (FIG. 1c). TNF treatment...
example ii
IKK-γ Binds the IKK-β Component of IKK and Associates with IKK-α in the Absence of IKK-β
[0116] This example demonstrates that IKK-γ directly binds IKK-β and that IKK-γ can form a complex with IKK-α in IKK-β deficient cells.
A. IKK-γ Interacts Physically with IKK-α / β
[0117] To confirm that the cloned IKK-γ protein interacts with IKK-α / β subunits in cells, an expression vector for N-terminally hemagglutinin (HA)-tagged IKK-γ was cotransfected into HeLa cells with expression vectors for either Flag-IKK-α or Flag-IKK-β. Transfected cell lysates were immunoprecipitated with anti-HA followed by immunoblotting with anti-Flag. As shown in FIG. 3a, IKK-γ interacted efficiently with IKK-α and IKK-β. Similar results were obtained when the immunoprecipitating antibody was directed to the Flag epitope and the immunecomplexes were immunoblotted with anti-HA antibody recognizing the epitope on IKK-γ. Immunoprecipitation of transiently expressed HA-IKK-γ resulted in isolation of endogenous IKK-αX (...
example iii
IKK-γ is an Essential Component of the IκB Kinase
[0124] This example uses antisense methodology to demonstrate that IKK-γ is an essential component of the IκB kinase (IKK).
A. IKK-γ is an Essential Component of the IκB Kinase
[0125] Immunoprecipitation of HA-IKK-γ from transiently transfected cells resulted in isolation of an IκB kinase activity that was stimulated by either TNF or IL-1 (FIG. 4a) as was seen when anti-IKK-α was used to isolate the IKK complex. These results were identical to those obtained when anti-IKK-α was used to isolated the IKK complex. This similarity was due to efficient interaction between transiently expressed HA-IKK-γ and other IKK components. Gel filtration analysis indicated that HA-IKK-γ was incorporated into the large 900 kDa IKK complex, precisely coeluting with IKK-α (FIG. 4b).
[0126] The role of IKK-γ in IKK activation was analyzed using expression of IKK-γ antisense constructs made in pcDNA3.1 / Myc-His (Invitrogen). Whereas cotransfection of an I...
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