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. 2006 Aug 15;398(1):63-71.
doi: 10.1042/BJ20051664.

Characterization of COMMD protein-protein interactions in NF-kappaB signalling

Affiliations

Characterization of COMMD protein-protein interactions in NF-kappaB signalling

Prim de Bie et al. Biochem J. .

Abstract

COMMD [copper metabolism gene MURR1 (mouse U2af1-rs1 region 1) domain] proteins constitute a recently identified family of NF-kappaB (nuclear factor kappaB)-inhibiting proteins, characterized by the presence of the COMM domain. In the present paper, we report detailed investigation of the role of this protein family, and specifically the role of the COMM domain, in NF-kappaB signalling through characterization of protein-protein interactions involving COMMD proteins. The small ubiquitously expressed COMMD6 consists primarily of the COMM domain. Therefore COMMD1 and COMMD6 were analysed further as prototype members of the COMMD protein family. Using specific antisera, interaction between endogenous COMMD1 and COMMD6 is described. This interaction was verified by independent techniques, appeared to be direct and could be detected throughout the whole cell, including the nucleus. Both proteins inhibit TNF (tumour necrosis factor)-induced NF-kappaB activation in a non-synergistic manner. Mutation of the amino acid residues Trp24 and Pro41 in the COMM domain of COMMD6 completely abolished the inhibitory effect of COMMD6 on TNF-induced NF-kappaB activation, but this was not accompanied by loss of interaction with COMMD1, COMMD6 or the NF-kappaB subunit RelA. In contrast with COMMD1, COMMD6 does not bind to IkappaBalpha (inhibitory kappaBalpha), indicating that both proteins inhibit NF-kappaB in an overlapping, but not completely similar, manner. Taken together, these data support the significance of COMMD protein-protein interactions and provide new mechanistic insight into the function of this protein family in NF-kappaB signalling.

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Figures

Figure 1
Figure 1. Genetic description of COMMD6
(A) Schematic representation of COMMD6 and the COMMD6 isoform a transcripts. Boxes indicate individual exons; exon sizes are indicated in bp. Sequences encoding the COMM domain are indicated in grey. (B) RT-PCR analysis of the expression of COMMD6, COMMD6 isoform a and COMMD1 in different human tissues. G3PDH (glyceraldehyde-3-phosphate dehydrogenase) RT-PCR was performed as positive control, and, as a negative control (NC), water was used as template. Sk., skeletal. (C) Alignment of COMMD6 amino acid sequences of different species. The COMM domain is underlined. Black, dark-grey or light-grey background colours indicate a 100%, 50% or 20% degree of conservation respectively. Arrows indicate residues mutated for experiments described in Figures 5 and 6.
Figure 2
Figure 2. Detection of endogenous COMMD1- and COMMD6-containing complexes
(A) Constructs encoding COMMD1–HA (upper panel) or COMMD6–FLAG (lower panel) were used in coupled in vitro transcription–translation reactions in the presence of radiolabelled amino acids. Labelled proteins were analysed directly (Input) or immunoprecipitated (IP) using pre-immune rabbit serum, COMMD1 antiserum, affinity-purified COMMD6 antiserum or anti-GST antibody. Proteins were separated by SDS/PAGE and visualized by fluorography. Apparent molecular-mass markers are indicated in kDa on the left. (B) Cell lysates of untransfected HEK-293 cells were directly analysed (Input) or used for immunoprecipitation (IP) using pre-immune rabbit serum, COMMD1 antiserum, COMMD6 antiserum or anti-GST antibody. Precipitates were rinsed and separated by SDS/PAGE and immunoblotted using antibodies as indicated. IgG light chain is indicated by *. Apparent molecular-mass markers are indicated in kDa on the left. (C) HEK-293 cells were transfected with cDNA constructs encoding COMMD6–FLAG, COMMD6 isoform a–FLAG, GST, COMMD1–GST or COMMD6–GST as indicated. Cell lysates were made which were used for glutathione–Sepharose precipitation. Precipitates were washed and separated by SDS/PAGE and immunoblotted as indicated. Input indicates direct analysis of cell lysates. Apparent molecular-mass markers are indicated in kDa on the left. WB, Western blot.
Figure 3
Figure 3. Interaction between COMMD1 and COMMD6 is direct and mediated through the COMM domain
(A) Constructs encoding the indicated proteins were used in coupled in vitro transcription–translation reactions in the presence of radiolabelled amino acids. Labelled proteins were analysed directly (Input; left panel) or immunoprecipitated using anti-FLAG antibody (IP: Flag; right panels). Proteins were separated by SDS/PAGE and visualized by fluorography. Apparent molecular-mass markers are indicated in kDa on the left. (B) YGH1 yeast was transformed with constructs encoding COMMD1 and COMMD6, fused to Gal4DBD (GAL4 DNA-binding domain) or Gal4AD (GAL4 activation domain) as indicated. All constructs were also transfected together with empty vectors as negative controls. Strains were grown in duplicate on selective medium and were subjected to a filter lift test for β-galactosidase activity. The interaction between ATOX1 (antioxidant protein 1) and ATP7B was used as positive control for β-galactosidase activity. (C) Different COMMD1 deletion constructs based on COMMD1 exon boundaries are depicted schematically. Numbers indicate predicted amino acid residues. All constructs were fused to the coding region of the Gal4DBD. YGH1 yeast were transformed with the COMMD1–Gal4DBD deletion constructs and COMMD6–Gal4AD as indicated. Strains were grown in duplicate on selective medium and were subjected to a filter lift test for β-galactosidase activity. The interaction between ATOX1 and ATP7B was used as positive control for β-galactosidase activity.
Figure 4
Figure 4. Bimolecular fluorescence complementation with COMMD1 and COMMD6
The principle of bimolecular fluorescence complementation is depicted schematically. HEK-293 cells were transfected with expression vectors encoding COMMD1–C-YFP and –N-YFP (A), COMMD6–N-YFP and –C-YFP (B), COMMD1-C-YFP and COMMD6-N-YFP (C), COMMD1–N-YFP and Smac/Diablo–C-YFP (D) or COMMD1–N-YFP and COMMD6–C-YFP (E). After overnight transfection, nuclear counterstain was performed by incubating cells with Hoechst 33342 before acquiring confocal images. The YFP images, Hoechst images and overlay images are shown as indicated.
Figure 5
Figure 5. COMMD6 inhibits NF-κB signalling through residues in the COMMD
HEK-293 cells were transfected with empty vector (A, B), or expression vectors encoding COMMD1–FLAG (A), COMMD6–FLAG (A, B), COMMD6–FLAG mutants W24A, P41A, W24A/P41A (B) or IκBα S.D. (A, B) along with a 2κB–luciferase reporter plasmid as indicated. Cells were incubated with (open bars) or without (closed bars) TNF and were lysed after 12 h. Upper panels: luciferase activities in the lysates were measured and expressed as fold induction relative to unstimulated empty-vector-transfected cells. Results are means±S.D. for three independent experiments performed in triplicate. Lower panels: immunoblotting of cell lysates using anti-FLAG antibodies was performed to confirm expression of COMMD1–FLAG and COMMD6–FLAG. Apparent molecular-mass markers are indicated in kDa on the left. WB, Western blot.
Figure 6
Figure 6. COMMD6–FLAG W24A/P41A mutant interacts with COMMD1, COMMD6 and RelA, but not with IκBα
(A and B) Glutathione–Sepharose precipitation using cell lysates of HEK-293 cells expressing the COMMD6–FLAG wild-type (WT) and point mutants and COMMD1–GST (A) or COMMD6–GST (B). Precipitates were washed and separated by SDS/PAGE and immunoblotted as indicated. Input indicates direct analysis of cell lysates. Apparent molecular-mass markers are indicated in kDa on the left. (C and D) Glutathione–Sepharose precipitation performed on the cell lysates of HEK-293 cells expressing COMMD1 and wild-type (WT) and mutant COMMD6 as GST fusion proteins, that were either untransfected (C) or transfected with an expression vector encoding HA–IκBα (D). Precipitates were washed and separated by SDS/PAGE and immunoblotted for RelA (C), HA (D) or GST (C and D) as indicated. Input indicates direct analysis of cell lysates. Apparent molecular-mass markers are indicated in kDa on the left. WB, Western blot.

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