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. Author manuscript; available in PMC: 2010 Oct 9.
Published in final edited form as: Mol Cell. 2009 Oct 9;36(1):131–140. doi: 10.1016/j.molcel.2009.07.025

Figure 3. KEAP1 functions as a CUL3-based E3 ligase of IKKβ.

Figure 3

(A) KEAP1-dependent K48 ubiquitination of IKKβ in vivo. Flag-tagged IKKβ and HA-tagged KEAP1 were cotransfected with either wild-type or K48R mutant ubiquitin into HEK-293T cells. After 24 hr post-transfection, cells were treated with MG132 for 6 hr. Flag-tagged IKKβ was immunoprecipitated, and then ubiquitination was analyzed by blotting with anti-ubiquitin antibody.

(B) KEAP1 bridges the interaction between IKKβ and the CUL3-RBX1 complex. CUL3 immunoprecipitates were analyzed by immunoblot with the indicated antibodies.

(C) In vitro ubiquitination of IKKβ by the KEAP1-CUL3-RBX1 complex. Flag-tagged IKKβ was incubated with KEAP1, CUL3, and RBX1 in the presence of E1, E2, His-Ubiquitin, and ATP as indicated.

(D) Human IKKβ contains a KEAP1-binding (D/N)XE(T/S)GE motif (D, aspartic acid; N, asparagine; E, glutamic acid; T, threonine; S, serine; G. glycine; and X, any amino acid).

(E) KEAP1 markedly decreased steady-state levels of wild-type but not E36A and E39A mutant IKKβ. GFP served as an internal control.

(F–G). E36A and E39A mutant IKKβ markedly reduced their binding ability to KEAP1 and were resistant to KEAP1-mediated ubiquitination.

(H) Mass spectrometry analysis revealed that IKKβ K555 is a polyuibiquitination site. Flag-tagged IKKβ, HA-tagged KEAP1, and HA-tagged ubiquitin were cotransfected into HEK-293T cells. After 1 day post-transfection, cells were treated with MG132 for 6hr to prevent the degradation of polyubiquitinated IKKβ. Polyubiquitinated IKKβ were pull down by using antibodies against HA tags and analyzed by μ-LC/MS/MS mass spectrometry.