NEMO specifically recognizes K63-linked poly-ubiquitin chains through a new bipartite ubiquitin-binding domain

Abstract

An important property of NEMO, the core element of the IKK complex involved in NF-kappaB activation, resides in its ability to specifically recognize poly-ubiquitin chains. A small domain called NOA/UBAN has been suggested to be responsible for this property. We recently demonstrated that the C-terminal Zinc Finger (ZF) of NEMO is also able to bind ubiquitin. We show here by ZF swapping and mutagenesis that this represents its only function. While neither NOA nor ZF shows any preference for K63-linked chains, we demonstrate that together they form a bipartite high-affinity K63-specific ubiquitin-binding domain. A similar domain can be found in two other proteins, Optineurin and ABIN2, and can be freely exchanged with that of NEMO without interfering with its activity. This suggests that the main function of the C-terminal half of NEMO is to specifically bind K63-linked poly-ubiquitin chains. We also demonstrate that the recently described binding of NEMO to linear poly-ubiquitin chains is dependent on the NOA alone and does not require the presence of the ZF.

Figure 1

Alignment of the C-terminal region (LZ-linker-ZF for NEMO) of human NEMO, Optineurin (Optn) and ABIN2. The NOA which is part of the LZ is indicated. Amino acids conserved in two proteins are indicated in green, and in red when conserved in the three proteins. The last amino acid indicated for each protein is the actual C-terminus.

Figure 2

NF-κB activity elicited by ZF-swap NEMO derivatives in NEMO-deficient T cells. NF-κB activation by TNF-α or PMA/ionomycin was measured in NEMO-deficient JM4.5.2 T cells following transfection of various NEMO derivatives (panels A and B represent separate series of experiments) together with a NF-κB-dependent firefly luciferase reporter construct and a control Renilla luciferase-expressing plasmid. The results shown were obtained from an experiment done in triplicate (see section Materials and methods). 100% corresponds to the ratio between stimulated and non-stimulated cells following transfection of WT NEMO; it corresponds to 9.6- and 6.8-fold for TNF-α and PMA/ionomycin, respectively. Each condition was repeated at least three times and gave similar results. The expression level of the different constructs was determined in a typical experiment by western blot analysis using an anti-HA antibody (shown below the graphs).

Figure 3

Pull-down activity of NEMO derivatives for K48- and K63-linked chains. HA-tagged NEMO derivatives were immunoprecipitated from transfected 293T cells and incubated with K48- or K63-linked poly-ubiquitin chains (3–7 in length). The interactions were set up as described under section Materials and methods; immunoprecipitated NEMO derivatives were incubated with poly-ubiquitin chains, washed and blotted for ubiquitin (WB: ubiquitin) and for HA-NEMO (WB: HA). K48 and K63 inputs correspond to 8% of the material used for the binding reactions. A non-specific band detected by the anti-ubiquitin antibody in the poly-ubiquitin preparations is shown by an asterisk. The dark background in lanes 9 and 11 is due to cellular ubiquitinated proteins co-precipitated with NEMO. Despite this background the NEMO-NOAZ ABIN2 and NEMO ZF-WRNIP1 maintain their specificity for K63-linked chains.

Figure 4

NF-κB activity elicited by NOAZ-swap NEMO derivatives in NEMO-deficient T cells (A) and MEFs (B). NF-κB activation by TNF-α or PMA/ionomycin was measured in NEMO-deficient JM4.5.2 T cells, and by TNF-α or LPS in NEMO-deficient MEFs, following transfection of various NEMO derivatives together with a NF-κB-dependent firefly luciferase reporter construct and a control Renilla luciferase-expressing plasmid. The results shown were obtained from an experiment performed in triplicate. 100% corresponds to the ratio between stimulated and non-stimulated cells following transfection of WT NEMO. It corresponds to 9.6- and 6.8-fold for TNF-α and PMA/ionomycin, respectively, in JM4.5.2 cells, and 9- and 7.7-fold for TNF-α and LPS, respectively, in NEMO-def MEFs. Each condition was repeated at least three times and gave similar results. The expression level of the different constructs was determined in a typical experiment by western blot analysis using an anti-HA antibody (shown below the graphs).

Figure 5

Pull-down activity of NEMO derivatives for linear tetra-ubiquitin chains. Pull-down experiments using HA-tagged NEMO derivatives and linear tetra-ubiquitin (GST-(Ub)4). The interactions were set up as described under section Materials and methods: cytosolic extracts of transfected 293T cells were incubated with GST(Ub)4 bound to glutathione agarose beads. After washing, the bound material was eluted with Laemmli buffer and blotted with anti-HA (middle panel). The level of expression of the NEMO derivatives was determined by a direct anti-HA blot (top panel). The level of recovered GST-(Ub)4 was determined by Ponceau staining of the membrane (lower panel).