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Negative regulation of NF-κB activity by brain-specific TRIpartite Motif protein 9.

Shi M, Cho H, Inn KS, Yang A, Zhao Z, Liang Q, Versteeg GA, Amini-Bavil-Olyaee S, Wong LY, Zlokovic BV, Park HS, García-Sastre A, Jung JU - Nat Commun (2014)

Bottom Line: The β-transducin repeat-containing protein (β-TrCP), a component of the Skp-Cullin-F-box-containing (SCF) E3 ubiquitin ligase complex, recognizes the NF-κB inhibitor IκBα and precursor p100 for proteasomal degradation and processing, respectively. β-TrCP thus plays a critical role in both canonical and non-canonical NF-κB activation.Consequently, expression or depletion of the TRIM9 gene significantly affected NF-κB-induced inflammatory cytokine production.This study not only elucidates a mechanism for TRIM9-mediated regulation of the β-TrCP SCF complex activity but also identifies TRIM9 as a brain-specific negative regulator of the NF-κB pro-inflammatory signalling pathway.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Microbiology and Immunology, Keck School of Medicine, University of Southern California, HMR Room 401, 2011 Zonal Avenue, Los Angeles, California 90033, USA.

ABSTRACT
The TRIpartite Motif (TRIM) family of RING-domain-containing proteins participate in a variety of cellular functions. The β-transducin repeat-containing protein (β-TrCP), a component of the Skp-Cullin-F-box-containing (SCF) E3 ubiquitin ligase complex, recognizes the NF-κB inhibitor IκBα and precursor p100 for proteasomal degradation and processing, respectively. β-TrCP thus plays a critical role in both canonical and non-canonical NF-κB activation. Here we report that TRIM9 is a negative regulator of NF-κB activation. Interaction between the phosphorylated degron motif of TRIM9 and the WD40 repeat region of β-TrCP prevented β-TrCP from binding its substrates, stabilizing IκBα and p100 and thereby blocking NF-κB activation. Consequently, expression or depletion of the TRIM9 gene significantly affected NF-κB-induced inflammatory cytokine production. This study not only elucidates a mechanism for TRIM9-mediated regulation of the β-TrCP SCF complex activity but also identifies TRIM9 as a brain-specific negative regulator of the NF-κB pro-inflammatory signalling pathway.

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TRIM9 competes with IκBα for β-TrCP binding and thereby blocks IκBα degradation(a) At 48h post transfection with Flag-β-TrCP and increasing amounts of HA-TRIM9 (µg) or HA-TRIM9 SA mutant, HEK293T cell lysates were used for IP with anti-Flag, followed by IB with anti-IκBα, anti-phospho-specific IκBα, or anti-HA antibody. HA-TRIM9 SA mutant was included as a control. (b) HEK293T-HA-TRIM9 or HA-SA mutant cells were treated with TNF-α and 25 µM MG132 for 1h and cell lysates were used for IP and IB with indicated antibodies. (c) HEK293T cells expressing Vector, HA-TRIM9 WT or the SA mutant were used for IP with anti-IκBα, followed by IB with anti-ubiquitin (Ubi) or anti-β-TrCP antibody. (d and e) SK-N-AS cells expressing HA-TRIM9 WT or the SA mutant (d) or carrying lentivirus containing scrambled shRNA (SC) or TRIM9-specific shRNA (KD) (e) were stimulated with IL-1 β for the indicated times, followed by IB with the indicated antibodies. The IκBα and Actin bands were quantified and the relative IκBα/Actin ratios were used to demonstrate the degradation rate of IκBα (bottom panel).The data are representative of three independent experiments.
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Figure 4: TRIM9 competes with IκBα for β-TrCP binding and thereby blocks IκBα degradation(a) At 48h post transfection with Flag-β-TrCP and increasing amounts of HA-TRIM9 (µg) or HA-TRIM9 SA mutant, HEK293T cell lysates were used for IP with anti-Flag, followed by IB with anti-IκBα, anti-phospho-specific IκBα, or anti-HA antibody. HA-TRIM9 SA mutant was included as a control. (b) HEK293T-HA-TRIM9 or HA-SA mutant cells were treated with TNF-α and 25 µM MG132 for 1h and cell lysates were used for IP and IB with indicated antibodies. (c) HEK293T cells expressing Vector, HA-TRIM9 WT or the SA mutant were used for IP with anti-IκBα, followed by IB with anti-ubiquitin (Ubi) or anti-β-TrCP antibody. (d and e) SK-N-AS cells expressing HA-TRIM9 WT or the SA mutant (d) or carrying lentivirus containing scrambled shRNA (SC) or TRIM9-specific shRNA (KD) (e) were stimulated with IL-1 β for the indicated times, followed by IB with the indicated antibodies. The IκBα and Actin bands were quantified and the relative IκBα/Actin ratios were used to demonstrate the degradation rate of IκBα (bottom panel).The data are representative of three independent experiments.

Mentions: To address whether TRIM9 competes with IκBα for β-TrCP binding, TRIM9, β-TrCP, and IκBα were expressed in HEK293T cells, followed by an examination of the interaction of β-TrCP with IκBα and TRIM9. This showed that TRIM9 WT, but not the TRIM9 SA mutant, impaired β-TrCP interaction with IκBα in a dosage-dependent manner (Fig. 4a). On the contrary, increased expression of IκBα did not significantly block the β-TrCP and TRIM9 interaction (Supplementary Fig 5). Similarly, when endogenous β-TrCP was immunopurified, its interaction with endogenous phosphorylated IκBα was also diminished in the presence of the TRIM9 WT, but not the SA mutant (Fig. 4b). Moreover, this reduction was more pronounced upon TNF-α stimulation. Indeed, expression of the TRIM9 WT suppressed the TNF-α -induced ubiquitination of IκBα, whereas expression of the SA mutant did not do so (Fig. 4c).


Negative regulation of NF-κB activity by brain-specific TRIpartite Motif protein 9.

Shi M, Cho H, Inn KS, Yang A, Zhao Z, Liang Q, Versteeg GA, Amini-Bavil-Olyaee S, Wong LY, Zlokovic BV, Park HS, García-Sastre A, Jung JU - Nat Commun (2014)

TRIM9 competes with IκBα for β-TrCP binding and thereby blocks IκBα degradation(a) At 48h post transfection with Flag-β-TrCP and increasing amounts of HA-TRIM9 (µg) or HA-TRIM9 SA mutant, HEK293T cell lysates were used for IP with anti-Flag, followed by IB with anti-IκBα, anti-phospho-specific IκBα, or anti-HA antibody. HA-TRIM9 SA mutant was included as a control. (b) HEK293T-HA-TRIM9 or HA-SA mutant cells were treated with TNF-α and 25 µM MG132 for 1h and cell lysates were used for IP and IB with indicated antibodies. (c) HEK293T cells expressing Vector, HA-TRIM9 WT or the SA mutant were used for IP with anti-IκBα, followed by IB with anti-ubiquitin (Ubi) or anti-β-TrCP antibody. (d and e) SK-N-AS cells expressing HA-TRIM9 WT or the SA mutant (d) or carrying lentivirus containing scrambled shRNA (SC) or TRIM9-specific shRNA (KD) (e) were stimulated with IL-1 β for the indicated times, followed by IB with the indicated antibodies. The IκBα and Actin bands were quantified and the relative IκBα/Actin ratios were used to demonstrate the degradation rate of IκBα (bottom panel).The data are representative of three independent experiments.
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Related In: Results  -  Collection

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Figure 4: TRIM9 competes with IκBα for β-TrCP binding and thereby blocks IκBα degradation(a) At 48h post transfection with Flag-β-TrCP and increasing amounts of HA-TRIM9 (µg) or HA-TRIM9 SA mutant, HEK293T cell lysates were used for IP with anti-Flag, followed by IB with anti-IκBα, anti-phospho-specific IκBα, or anti-HA antibody. HA-TRIM9 SA mutant was included as a control. (b) HEK293T-HA-TRIM9 or HA-SA mutant cells were treated with TNF-α and 25 µM MG132 for 1h and cell lysates were used for IP and IB with indicated antibodies. (c) HEK293T cells expressing Vector, HA-TRIM9 WT or the SA mutant were used for IP with anti-IκBα, followed by IB with anti-ubiquitin (Ubi) or anti-β-TrCP antibody. (d and e) SK-N-AS cells expressing HA-TRIM9 WT or the SA mutant (d) or carrying lentivirus containing scrambled shRNA (SC) or TRIM9-specific shRNA (KD) (e) were stimulated with IL-1 β for the indicated times, followed by IB with the indicated antibodies. The IκBα and Actin bands were quantified and the relative IκBα/Actin ratios were used to demonstrate the degradation rate of IκBα (bottom panel).The data are representative of three independent experiments.
Mentions: To address whether TRIM9 competes with IκBα for β-TrCP binding, TRIM9, β-TrCP, and IκBα were expressed in HEK293T cells, followed by an examination of the interaction of β-TrCP with IκBα and TRIM9. This showed that TRIM9 WT, but not the TRIM9 SA mutant, impaired β-TrCP interaction with IκBα in a dosage-dependent manner (Fig. 4a). On the contrary, increased expression of IκBα did not significantly block the β-TrCP and TRIM9 interaction (Supplementary Fig 5). Similarly, when endogenous β-TrCP was immunopurified, its interaction with endogenous phosphorylated IκBα was also diminished in the presence of the TRIM9 WT, but not the SA mutant (Fig. 4b). Moreover, this reduction was more pronounced upon TNF-α stimulation. Indeed, expression of the TRIM9 WT suppressed the TNF-α -induced ubiquitination of IκBα, whereas expression of the SA mutant did not do so (Fig. 4c).

Bottom Line: The β-transducin repeat-containing protein (β-TrCP), a component of the Skp-Cullin-F-box-containing (SCF) E3 ubiquitin ligase complex, recognizes the NF-κB inhibitor IκBα and precursor p100 for proteasomal degradation and processing, respectively. β-TrCP thus plays a critical role in both canonical and non-canonical NF-κB activation.Consequently, expression or depletion of the TRIM9 gene significantly affected NF-κB-induced inflammatory cytokine production.This study not only elucidates a mechanism for TRIM9-mediated regulation of the β-TrCP SCF complex activity but also identifies TRIM9 as a brain-specific negative regulator of the NF-κB pro-inflammatory signalling pathway.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Microbiology and Immunology, Keck School of Medicine, University of Southern California, HMR Room 401, 2011 Zonal Avenue, Los Angeles, California 90033, USA.

ABSTRACT
The TRIpartite Motif (TRIM) family of RING-domain-containing proteins participate in a variety of cellular functions. The β-transducin repeat-containing protein (β-TrCP), a component of the Skp-Cullin-F-box-containing (SCF) E3 ubiquitin ligase complex, recognizes the NF-κB inhibitor IκBα and precursor p100 for proteasomal degradation and processing, respectively. β-TrCP thus plays a critical role in both canonical and non-canonical NF-κB activation. Here we report that TRIM9 is a negative regulator of NF-κB activation. Interaction between the phosphorylated degron motif of TRIM9 and the WD40 repeat region of β-TrCP prevented β-TrCP from binding its substrates, stabilizing IκBα and p100 and thereby blocking NF-κB activation. Consequently, expression or depletion of the TRIM9 gene significantly affected NF-κB-induced inflammatory cytokine production. This study not only elucidates a mechanism for TRIM9-mediated regulation of the β-TrCP SCF complex activity but also identifies TRIM9 as a brain-specific negative regulator of the NF-κB pro-inflammatory signalling pathway.

Show MeSH
Related in: MedlinePlus