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. 2014 Aug 1;461(3):531-7.
doi: 10.1042/BJ20140444.

An unexpected twist to the activation of IKKβ: TAK1 primes IKKβ for activation by autophosphorylation

Jiazhen Zhang  1 , Kristopher Clark  1 , Toby Lawrence  2 , Mark W Peggie  1 , Philip Cohen  1
Affiliations

An unexpected twist to the activation of IKKβ: TAK1 primes IKKβ for activation by autophosphorylation

Jiazhen Zhang et al. Biochem J. .

Abstract

IKKβ {IκB [inhibitor of NF-κB (nuclear factor κB)] kinase β} is required to activate the transcription factor NF-κB, but how IKKβ itself is activated in vivo is still unclear. It was found to require phosphorylation by one or more 'upstream' protein kinases in some reports, but by autophosphorylation in others. In the present study, we resolve this contro-versy by demonstrating that the activation of IKKβ induced by IL-1 (interleukin-1) or TNF (tumour necrosis factor) in embryonic fibroblasts, or by ligands that activate Toll-like receptors in macrophages, requires two distinct phosphorylation events: first, the TAK1 [TGFβ (transforming growth factor β)-activated kinase-1]-catalysed phosphorylation of Ser¹⁷⁷ and, secondly, the IKKβ-catalysed autophosphorylation of Ser¹⁸¹. The phosphorylation of Ser¹⁷⁷ by TAK1 is a priming event required for the subsequent autophosphorylation of Ser¹⁸¹, which enables IKKβ to phosphorylate exogenous substrates. We also provide genetic evidence which indicates that the IL-1-stimulated, LUBAC (linear ubiquitin chain assembly complex)-catalysed formation of linear ubiquitin chains and their interaction with the NEMO (NF-κB essential modulator) component of the canonical IKK complex permits the TAK1-catalysed priming phosphorylation of IKKβ at Ser¹⁷⁷ and IKKα at Ser¹⁷⁶. These findings may be of general significance for the activation of other protein kinases.

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Figures

Figure 1
Figure 1. Effect of protein kinase inhibitors on the phosphorylation of IKKβ at Ser177 and/or Ser181 in MEFs from IKKα-deficient mice and BMDMs from knockin mice expressing catalytically inactive IKKα[S176A/S180A]
(A) MEFs from IKKα-knockout mice were incubated for 1 h without (−) or with (+) 1.0 μM NG25, 1.0 μM 5Z-7-oxozeaenol or 5.0 μM BI605906, then stimulated for 10 min with 5.0 ng/ml IL-1. Cell lysates were subjected to SDS/PAGE and immunoblotting as described in the Experimental section. (B) The experiment was performed exactly as in (A) except that the cells were stimulated with 10 ng/ml TNF. (C and D) BMDMs from knockin mice expressing the catalytically inactive IKKα[S176A/S180A] mutant were incubated for 1 h without (−) or with (+) 2 μM NG25 or 2 μM BI605906, then stimulated for 10 min with 1 μg/ml Pam3CSK4 (C) or 0.1 μg/ml LPS (D). Cell extracts were subjected to SDS/PAGE and immunoblotted as in (A and B).
Figure 2
Figure 2. Expression of IKKβ[S177E] induces the autophosphorylation of Ser181 and activation of IKKβ
(A) MEFs from IKKα-deficient mice stably expressing HA-tagged wild-type IKKβ (WT), IKKβ[S177E] (S177E) or IKKβ[S177A] (S177A) or empty vector (EV) were incubated for 16 h with 1.0 μg/ml (WT and EV), 0.2 μg/ml (S177A) or 0.1 μg/ml (S177E) doxycycline to induce the expression of these proteins, and then for 1 h without (−) or with (+) 5 μM NG25 or 5 μM BI605906 and lysed. Extract [20 μg (EV, S177E and S177A) or 80 μg (WT) protein] were analysed by immunoblotting with the antibodies indicated. (B) As in (A), except that Ser181 phosphorylation was studied in MEFs stably expressing HA–IKKβ[D166A/S177E] and HA–IKKβ[S177E], and no inhibitors were present. (C and D) HA-tagged IKKβ[S177E] was transfected into HEK-293 cells, immunoprecipitated from the cell extracts, incubated without (−) or with (+) PP1γ and assayed for IKKβ activity (C) or immunoblotted with antibodies that recognize IKKβ phosphorylated at Ser181 or all forms of IKKβ (D). The results in (C) are means±S.E.M. of duplicate determinations. Similar results were obtained in two other independent experiments.
Figure 3
Figure 3. IKKβ phosphorylated at Ser177 has little activity if Ser181 is not phosphorylated
(A) MEFs from IKKα-deficient mice were incubated for 1 h without (−) or with (+) 5.0 μM BI 605906 or 2 μM NG25, then stimulated for 10 min with 5.0 ng/ml IL-1. The endogenous IKKβ was immunoprecipitated from 0.2 mg of cell extract protein and assayed for activity. (B) The immunoprecipitates from (A) were denatured in SDS before and after the assay of IKKβ, and aliquots of each sample were subjected to SDS/PAGE, transferred on to PVDF membranes and immunoblotted with antibodies that recognize IKKβ phosphorylated at Ser177 or Ser181 or all forms of IKKβ.
Figure 4
Figure 4. Met1-linked ubiquitin chains and their interaction with NEMO are required for the IL-1-stimulated phosphorylation of IKKα and IKKβ in MEFs
(A) Cells from wild-type (HOIP[WT]) or knockin mice expressing the HOIP[C879S] mutant were stimulated with 5 ng/ml IL-1 for the times indicated and lysed. The extract (20 μg of protein) was subjected to immunoblotting and probed with the antibodies indicated. (B) As in (A) except that MEFs from NEMO[D311N]-knockin and wild-type mice were used.
Figure 5
Figure 5. Proposed mechanism for the activation of IKKβ by IL-1 in IKKα-deficient MEFs
IL-1 stimulates the formation of hybrid ubiquitin chains in which LUBAC-generated Met1-linked ubiquitin oligomers are attached covalently to TRAF6-generated Lys63-linked ubiquitin oligomers. The binding of Lys63-linked ubiquitin to TAB2 or TAB3 activates the TAK1 complex by inducing autophosphorylation of the catalytic subunit at Thr187 [31]. The M1-linked ubiquitin chains interact with NEMO permitting TAK1 to phosphorylate IKKβ at Ser177. Phosphorylation of Ser177 allows autophosphorylation of Ser181. The activated IKKβ can then phosphorylate IκBα. Sites of phosphorylation are denoted by ‘P’.
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