CSN controls NF-kappaB by deubiquitinylation of IkappaBalpha - PubMed (original) (raw)

Figure 1

CSN associates with IκBα. (A) Cells were transiently transfected with a FlagCSN2 expression plasmid (lanes 2–11) to enhance CSN assembly. After 20 h, cells were stimulated with TNFα for the indicated periods of time (lanes 4–11) or left untreated (lanes 1–3). At 1 h before TNFα stimulation, cells were treated with either MG132 (lanes 3, 5, 7, 9 and 11) or vehicle (DMSO, lanes 1, 2, 4, 6, 8 and 10). After immunoprecipitation of endogenous IκBα from total lysates, interaction of the CSN with IκBα was detected with an anti-Flag antibody that recognises FlagCSN2 within the CSN (upper panel). The stripped immunoblot was developed with an anti-IκBα antibody to verify TNFα-induced turnover of IκBα, which is inhibited by MG132 (second panel). Ectopic expression of FlagCSN2 was detected in cell lysates using an anti-Flag antibody. Immunodetection of ERK was performed as a protein load control. (B) As a supplement for the experiment shown in (A), only samples obtained from cells not treated with MG132 were analysed. Ectopic expression of FlagCSN2 was detected using an anti-Flag antibody. Overexpression of FlagCSN2 enhanced CSN assembly, as shown in an immunoblot using an antibody against endogenous CSN7.

Figure 2

Knockdown of CSN2 affects the basal protein level of IκBα. Cells were transiently transfected with one of two different CSN2-specific siRNAs or transfected with a nontargeting siRNA (mock). After 48 h, they were harvested by RIPA lysis. (A) Cell lysates from mock-transfected and CSN2-knockdown cells were analysed by SDS–PAGE and Western blotting. Immunodetection of CSN subunits, Cul1, IκBα, β-TrCP and ERK (see indicated panels), was performed using specific antibodies. (B) Quantification of the results shown for β-TrCP and IκBα in (A). Densitometric values for protein bands were normalised to ERK content in each sample and depicted relative to the values of untreated mock-transfected cells. The data are representative for at least three independent experiments.

Figure 3

Knockdown of the CSN affects the inducible turnover of IκBα and nuclear translocation of NF-κB. Cells were transiently transfected with CSN2siRNA1 or CSN5siRNA. After 48 h, cells were stimulated for different periods of time, as indicated, with TNFα. The cells were harvested in hypotonic lysis buffer, followed by cytosolic/nuclear fractionation. Efficient knockdown of CSN2 or CSN5 (indicated panels) was analysed in cell lysates by immunodetection. In order to control the purity of cytosolic and nuclear preparations, ERK was immunodetected as a cytosolic and HDAC1 as a nuclear marker protein. (A) Cytosolic fractions of CSN2-knockdown cells were analysed by SDS–PAGE and Western blotting. To confirm TNFα-induced phosphorylation and turnover of IκBα, immunodetection of IκBα and its Ser32/Ser36-phosphorylated form was performed using specific antibodies. The p65 and p50 subunits (see the indicated panels) were immunodetected to show their predominant cytosolic localisation. (B) Quantification of the results shown for IκBα in (A) was performed as described in Figure 2B. (C) Endogenous IκBα was immunoprecipitated from cytosolic fractions of HeLa cells, transfected with CSN2siRNA1 or a nontargeting siRNA. Immunoprecipitates were analysed by SDS–PAGE and Western blotting. The IκBα protein level and its multiple high-molecular-weight forms of phosphorylated IκBα were immunodetected using IκBα- and phospho-IκBα-specific antibodies, respectively. (D) Nuclear fractions were analysed by SDS–PAGE and Western blotting. Translocated p65, Ser536-phosphorylated p65, as well as p50 (indicated panels) were immunodetected using specific antibodies. (E) Quantification of the results shown for p50 and p65 in (D). Densitometric values for protein bands were normalised to HDAC1 content in each sample and depicted relative to the values of untreated mock-transfected cells. (F) Nuclear fractions were analysed by Electrophoretic mobility shift assay (EMSA). The shifted DNA-bound NF-κB p50/p65 heterodimer and the shifted DNA-bound Oct-1 transcription factor (used as a load control) are indicated. To test the specificity, competition (using 10 and 20 ng of the nonlabelled double-stranded Igκ oligonucleotide) and antibody supershifting using anti-p50 and anti-p65 antibodies as well as preimmune serum were performed. (G) Cytosolic fractions of CSN5-knockdown cells were analysed by SDS–PAGE and Western blotting. To confirm TNFα-induced phosphorylation and turnover of IκBα, immunodetection of IκBα and its Ser32/Ser36-phosphorylated form was performed using specific antibodies. Neddylated Cul1 (Cul1-Nedd8) was immunodetected, using a Cul1-specific antibody. (H) Quantification of the results shown for IκBα in (G) was performed as described in Figure 2B.

Figure 4

CSN regulates CRL assembly. (A) Cells were transiently transfected with CSN2siRNA1 to functionally disrupt the CSN. At 48 h after transfection, mock-transfected and CSN2-knockdown cells were stimulated for different periods of time with TNFα or left untreated, as indicated. The cells were harvested in hypotonic lysis buffer followed by cytosolic/nuclear fractionation. Endogenous β-TrCP was immunoprecipitated from the cytosolic fractions and the immunoprecipitates analysed by SDS–PAGE and Western blotting. Cul1 associated with β-TrCP as well as neddylated Cul1 (Cul1-Nedd8) was immunodetected, using Cul1- or Nedd8-specific antibodies, as indicated. Reduced expression of immunodetected β-TrCP and Cul1 in lysates of CSN2siRNA1-treated cells was shown by use of protein-specific antibodies. IκBα and phosphorylated IκBα were immunodetected using specific antibodies. Immunodetection of CSN2 was shown as a control for efficient knockdown. Equal amounts of protein in the lysates were controlled by immunodetection of ERK (indicated panels). (B) Endogenous Nedd8 was immunoprecipitated from cytosolic fractions of HeLa cells, transfected with CSN2siRNA1 or a nontargeting siRNA. Immunoprecipitates from mock-transfected and CSN2siRNA1-treated cells were analysed by SDS–PAGE and Western blotting. Associated β-TrCP as well as Cul1 was detected using β-TrCP- and Cul1-specific antibodies, respectively. The neddylation state of Cul1 was immunodetected in the total cell lysate using a Cul1-specific antibody. To verify the functional impairment of CSN, hyperneddylation and a decrease in Cul1 expression, as well as increased expression of IκBα in CSN2-knockdown versus mock-transfected cells was immunodetected in the cell lysates using Cul1- or IκBα-specific antibodies, as indicated. ERK was immunodetected to show equal amounts of protein in the cell lysates.

Figure 5

CSN promotes deubiquitinylation of IκBα. (A) Cells were either transiently transfected with CSN2siRNA1 (lanes 2, 4, 6, 8, 10, 12, 14 and 16) or mock transfectd (lanes 1, 3, 5, 7, 9, 11, 13 and 15). Then, cells were stimulated for different periods of time with TNFα or left untreated as indicated. At 1 h before stimulation, MG132 (lanes 3, 4, 7, 8, 11, 12, 15 and 16) or vehicle (DMSO, lanes 1, 2, 5, 6, 9, 10, 13 and 14), was added to the culture medium. After harvest of the cells by RIPA lysis, endogenous IκBα was immunoprecipitated, using an anti-IκBα antibody. Ubiquitinylated IκBα was immunodetected with an anti-ubiquitin antibody. The blot was stripped and developed with an anti-IκBα antibody to show TNFα-induced degradation and re-accumulation of IκBα. In addition, CSN2 was immunodetected in the cell lysates, using an anti-CSN2 antibody, to verify efficient knockdown of the CSN2 protein. Immunodetection of ERK in the cell lysates was performed as protein load control. (B) T7IκBα was expressed either alone (lanes 2–9) or in combination with FlagCSN2 (lanes 11–18) by transient transfection of equal amounts of cDNA in HeLa cells. Before cell lysis, the cells were stimulated with TNFα (lanes 4–9 and 13–18) for the indicated periods of time or left untreated (lanes 1–3 and 10–12). Additionally, the cells were treated for 1 h with either MG132 (lanes 3, 5, 7 9, 12, 14, 16 and 18) or vehicle (DMSO, lanes 1, 2, 4, 6, 8, 10, 11, 13, 15 and 17) before stimulation. Ectopically expressed T7IκBα was immunoprecipitated from cell lysates using an anti-T7 antibody and the protein complexes were subjected to SDS–PAGE and Western blotting. T7IκBα ubiquitinylation in TNFα-stimulated cells was determined by immunostaining with an anti-ubiquitin antibody (upper panels). The stripped immunoblots were developed with an anti-IκBα antibody to detect the TNFα-induced proteolytic turnover of T7IκBα, which was inhibited by MG132 (second panels). Ectopic expression of FlagCSN2, which mediates enhanced expression of CSN, was recognised by immunodetection with an anti-Flag antibody in the total cell lysate. Immunodetection of ERK was performed as protein load control.

Figure 6

CSN-associated USP15 deubiquitinylates IκBα. (A) Cells were either transiently transfected with USP15siRNA or mock transfected. After 48 h, cells were stimulated for different periods of time, as indicated, with TNFα. Cytosolic fractions of USP15-knockdown cells were analysed by SDS–PAGE and Western blotting. To confirm TNFα-induced phosphorylation and turnover of IκBα, immunodetection of IκBα and its Ser32/Ser36-phosphorylated form was performed using specific antibodies. USP15 was immunodetected to confirm knockdown efficiency. Immunodetection of ERK in the cell lysates was performed as protein load control. (B) Quantification of the results shown for IκBα in (A) was performed as described in Figure 2B. (C) Nuclear fractions were analysed by EMSA as described in Figure 3F. (D) Cells were either transiently transfected with USP15siRNA (lanes 2, 4, 6, 8, 10, 12, 14, 16, 18 and 20) or mock-transfected (lanes 1, 3, 5, 7, 9, 11, 13, 15, 17 and 19). Then, cells were stimulated for different periods of time with TNFα (lanes 5–20) or left untreated (lanes 1–4). At 1 h before stimulation, MG132 (lanes 3, 4, 7, 8, 11, 12, 15, 16, 19 and 20) or vehicle (DMSO, lanes 1, 2, 5, 6, 9, 10, 13, 14, 17 and 18) was added to the culture medium. After harvest of the cells, endogenous IκBα was immunoprecipitated from cytosolic fractions of HeLa cells using an anti-IκBα antibody. Immunoprecipitates were analysed by SDS–PAGE and Western blotting. The IκBα protein and its multiple high-molecular-weight forms of phosphorylated IκBα were immunodetected using IκBα- and phospho-IκBα-specific antibodies. Ubiquitinylated IκBα was immunodetected with an anti-ubiquitin antibody. USP15 was immunodetected in the cell lysates, using an anti-USP15 antibody, to verify efficient knockdown of the USP15 protein. Immunodetection of ERK in the cell lysates was performed as protein load control. (E) Cells were preincubated for 1 h (lanes 2 and 4–6) with MG132 or treated with vehicle (DMSO, lanes 1 and 3) and subsequently stimulated (lanes 3–6) or not (lanes 1 and 2) with TNFα for 15 min. IκBα immunoprecipitates were incubated for 4 h at 37°C in the presence of an ATP regenerating system. In addition, purified CSN (lane 5) or recombinant USP15 (lane 6) was added to catalyse deubiquitinylation of IκBα. Aliquots of the reactions were analysed by SDS–PAGE and Western blotting. Immunodetection of ubiquitin, IκBα and its Ser32/Ser36-phosphorylated form, CSN subunits and USP15 (see indicated panels) was performed using specific antibodies. Immunodetection of ERK in the cell lysates was used as a control for equal amounts of protein applied to each immunoprecipitation.

Figure 7

Model of CSN-mediated IκBα control. (A) In stimulated cells, the activated IKK complex phosphorylates Ser32 and Ser36 in the N-terminus of IκBα. When phosphorylated, these serine residues are part of a ‘degron' that is specifically recognised by β-TrCP, the CRL substrate adapter for IκBα. CRL activity is promoted by neddylation of Cul1. The latter facilitates the recruitment of a ubiquitin-loaded ubiquitin-conjugating enzyme (E2) to the ROC1 subunit of the CRL, which then cooperates with the CRL to ubiquitinylate IκBα. (B) Ubiquitinylated IκBα is degraded via the 26S proteasome releasing the previously bound and inactivated NF-κB. (C) Released NF-κB translocates to the nucleus to activate target genes, including the _I_κ_B_α gene. As part of a negative feedback loop, re-accumulating IκBα can dissociate DNA-bound NF-κB and re-transport it to the cytosol. (D) During persistent stimulation, re-accumulated IκBα becomes phosphorylated in the cytosol by the IKK complex, thus initiating the cycle again. However, the CSN can enhance the stability of IκBα and thereby contribute to shut down NF-κB activity. This might be brought about in part by cycles of CSN5-mediated deneddylation of Cul1, which controls CRL activity. Most importantly, however, IκBα is deubiquitinylated by the CSN-associated DUB USP15. (E) Re-accumulated IκBα firmly associates with NF-κB, keeping it inactive in the cytosol.