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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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Related in: MedlinePlus

TRIM9 interacts with β-TrCP in a phosphorylation dependent manner(a) Immunopurified HA-TRIM9 from HEK293T cells was treated with or without λ-phosphatase (500U) for 30 min at 37C. 10% of untreated or λ-phosphatase-treated HA-TRIM9 protein was used for IB with anti-HA or anti-phospho-serine-specific antibody (p-Ser). The remaining untreated or λ-phosphatase-treated HA-TRIM9 protein was mixed with HEK293T cell lysates containing Flag-β -TrCP for 2h at 4°C, washed several times with 1% Tween-PBS washing buffer and subjected to IB with anti-Flag. Data are representative of at least three independent experiments. (b) E. coli BL21ΔSerB strain was engineered with tRNASep (derived from M. jannaschii tRNACys), SerRS (Sep-tRNA synthetase derived from M. maripaludis SepRS) and EF-Sep (Engineered phosphoserine specific EF-Tu). Italicized codon of the TRIM9 mRNA indicates the mutation site (ACG→UAG). Sep: Phosphoserine. (c and d) In vitro binding assay of bacterially purified HA-TRIM9 WT, the Ser76 phosphorylated (pS76) form or the Ser76-to-alanine (SA) mutated form with mammalian Flag-β-TrCP purified from 293T cells. IP with anti-HA and IB with anti-Flag (c) and vise versa (d).(e and f) 293T-TRIM9-V5 cells were stimulated with TNF-α for the indicated times (e) or pretreated with the indicated inhibitors (f), followed by TNF-α stimulation. Cell lysates were used for IP and IB with the indicated antibodies. DM (DMSO control), Fos (Fostriecin, PP2A, 4, 5 inhibitor, 100µM for 6hrs), FK (FK-506. PP2B inhibitor, 100µM for 6hrs), San (Sanguinarine, PP2C inhibitor, 10mM for 6hrs), Cal (Calyculin A, PP1, 2A, 4, 5 inhibitor, 10nM for 1hr). Ct Ig: isotype control immunoglobulin. (g) At 48 hr post-infection with lentivirus containing scrambled shRNA (SC) or TRIM9-specific shRNA (KD), 293T cells expressing V5-TRIM9 were used for IP with anti-V5, followed by IB with the indicated antibodies. The data are representative of three independent experiments.
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Figure 3: TRIM9 interacts with β-TrCP in a phosphorylation dependent manner(a) Immunopurified HA-TRIM9 from HEK293T cells was treated with or without λ-phosphatase (500U) for 30 min at 37C. 10% of untreated or λ-phosphatase-treated HA-TRIM9 protein was used for IB with anti-HA or anti-phospho-serine-specific antibody (p-Ser). The remaining untreated or λ-phosphatase-treated HA-TRIM9 protein was mixed with HEK293T cell lysates containing Flag-β -TrCP for 2h at 4°C, washed several times with 1% Tween-PBS washing buffer and subjected to IB with anti-Flag. Data are representative of at least three independent experiments. (b) E. coli BL21ΔSerB strain was engineered with tRNASep (derived from M. jannaschii tRNACys), SerRS (Sep-tRNA synthetase derived from M. maripaludis SepRS) and EF-Sep (Engineered phosphoserine specific EF-Tu). Italicized codon of the TRIM9 mRNA indicates the mutation site (ACG→UAG). Sep: Phosphoserine. (c and d) In vitro binding assay of bacterially purified HA-TRIM9 WT, the Ser76 phosphorylated (pS76) form or the Ser76-to-alanine (SA) mutated form with mammalian Flag-β-TrCP purified from 293T cells. IP with anti-HA and IB with anti-Flag (c) and vise versa (d).(e and f) 293T-TRIM9-V5 cells were stimulated with TNF-α for the indicated times (e) or pretreated with the indicated inhibitors (f), followed by TNF-α stimulation. Cell lysates were used for IP and IB with the indicated antibodies. DM (DMSO control), Fos (Fostriecin, PP2A, 4, 5 inhibitor, 100µM for 6hrs), FK (FK-506. PP2B inhibitor, 100µM for 6hrs), San (Sanguinarine, PP2C inhibitor, 10mM for 6hrs), Cal (Calyculin A, PP1, 2A, 4, 5 inhibitor, 10nM for 1hr). Ct Ig: isotype control immunoglobulin. (g) At 48 hr post-infection with lentivirus containing scrambled shRNA (SC) or TRIM9-specific shRNA (KD), 293T cells expressing V5-TRIM9 were used for IP with anti-V5, followed by IB with the indicated antibodies. The data are representative of three independent experiments.

Mentions: To test whether serine phosphorylation of the TRIM9 degron motif is required for its interaction with β-TrCP, immunopurified HA-TRIM9 complexes were treated with mock or λ-phosphatase, incubated with lysates containing Flag-β-TrCP, and then subjected to immunoblotting with anti-Flag antibody. This showed that treatment of immunopurified TRIM9 with λ-phosphatase drastically abrogated its β-TrCP-binding ability (Fig. 3a). Phosphoserine (Sep), the most abundant phosphoamino acid in the eukaryotic phosphoproteome, is not encoded in the genetic code, but is synthesized post translationally. Since our attempts to generate the pS76pS80 phospho-specific TRIM9 antibodies failed twice, we utilized the specific cotranslational Sep incorporation system38, 39to study the Ser76 residue of TRIM9. An Escherichia coli strain was genetically engineered to harbor a Sep-accepting transfer RNA (tRNASep), its cognate Sep–tRNAsynthetase (SepRS), and an engineered EF-Tu (EF-Sep) of Methanocaldococcus jannaschii, and then transformed with bacterial expression vector containing the N-terminal HA-tagged RING domain (1-160aa) of TRIM9 WT, the SA mutant, and the non-natural Sep-incorporated pS76(directed by UAG) mutant (Fig. 3b). These HA-TRIM9 proteins were purified from genetically engineered E. coli and used for in vitro pulldown (PD) assay with Flag-β-TrCP. This showed that the pS76 TRIM9 protein specifically and efficiently bound β-TrCP in vitro, whereas the WT and SA TRIM9 proteins did not bind (Fig. 3c, d), suggesting that the serine phosphorylation of the TRIM9 degron motif is necessary for binding the WD40 domain of β-TrCP.


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 interacts with β-TrCP in a phosphorylation dependent manner(a) Immunopurified HA-TRIM9 from HEK293T cells was treated with or without λ-phosphatase (500U) for 30 min at 37C. 10% of untreated or λ-phosphatase-treated HA-TRIM9 protein was used for IB with anti-HA or anti-phospho-serine-specific antibody (p-Ser). The remaining untreated or λ-phosphatase-treated HA-TRIM9 protein was mixed with HEK293T cell lysates containing Flag-β -TrCP for 2h at 4°C, washed several times with 1% Tween-PBS washing buffer and subjected to IB with anti-Flag. Data are representative of at least three independent experiments. (b) E. coli BL21ΔSerB strain was engineered with tRNASep (derived from M. jannaschii tRNACys), SerRS (Sep-tRNA synthetase derived from M. maripaludis SepRS) and EF-Sep (Engineered phosphoserine specific EF-Tu). Italicized codon of the TRIM9 mRNA indicates the mutation site (ACG→UAG). Sep: Phosphoserine. (c and d) In vitro binding assay of bacterially purified HA-TRIM9 WT, the Ser76 phosphorylated (pS76) form or the Ser76-to-alanine (SA) mutated form with mammalian Flag-β-TrCP purified from 293T cells. IP with anti-HA and IB with anti-Flag (c) and vise versa (d).(e and f) 293T-TRIM9-V5 cells were stimulated with TNF-α for the indicated times (e) or pretreated with the indicated inhibitors (f), followed by TNF-α stimulation. Cell lysates were used for IP and IB with the indicated antibodies. DM (DMSO control), Fos (Fostriecin, PP2A, 4, 5 inhibitor, 100µM for 6hrs), FK (FK-506. PP2B inhibitor, 100µM for 6hrs), San (Sanguinarine, PP2C inhibitor, 10mM for 6hrs), Cal (Calyculin A, PP1, 2A, 4, 5 inhibitor, 10nM for 1hr). Ct Ig: isotype control immunoglobulin. (g) At 48 hr post-infection with lentivirus containing scrambled shRNA (SC) or TRIM9-specific shRNA (KD), 293T cells expressing V5-TRIM9 were used for IP with anti-V5, followed by IB with the indicated antibodies. The data are representative of three independent experiments.
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Related In: Results  -  Collection

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Figure 3: TRIM9 interacts with β-TrCP in a phosphorylation dependent manner(a) Immunopurified HA-TRIM9 from HEK293T cells was treated with or without λ-phosphatase (500U) for 30 min at 37C. 10% of untreated or λ-phosphatase-treated HA-TRIM9 protein was used for IB with anti-HA or anti-phospho-serine-specific antibody (p-Ser). The remaining untreated or λ-phosphatase-treated HA-TRIM9 protein was mixed with HEK293T cell lysates containing Flag-β -TrCP for 2h at 4°C, washed several times with 1% Tween-PBS washing buffer and subjected to IB with anti-Flag. Data are representative of at least three independent experiments. (b) E. coli BL21ΔSerB strain was engineered with tRNASep (derived from M. jannaschii tRNACys), SerRS (Sep-tRNA synthetase derived from M. maripaludis SepRS) and EF-Sep (Engineered phosphoserine specific EF-Tu). Italicized codon of the TRIM9 mRNA indicates the mutation site (ACG→UAG). Sep: Phosphoserine. (c and d) In vitro binding assay of bacterially purified HA-TRIM9 WT, the Ser76 phosphorylated (pS76) form or the Ser76-to-alanine (SA) mutated form with mammalian Flag-β-TrCP purified from 293T cells. IP with anti-HA and IB with anti-Flag (c) and vise versa (d).(e and f) 293T-TRIM9-V5 cells were stimulated with TNF-α for the indicated times (e) or pretreated with the indicated inhibitors (f), followed by TNF-α stimulation. Cell lysates were used for IP and IB with the indicated antibodies. DM (DMSO control), Fos (Fostriecin, PP2A, 4, 5 inhibitor, 100µM for 6hrs), FK (FK-506. PP2B inhibitor, 100µM for 6hrs), San (Sanguinarine, PP2C inhibitor, 10mM for 6hrs), Cal (Calyculin A, PP1, 2A, 4, 5 inhibitor, 10nM for 1hr). Ct Ig: isotype control immunoglobulin. (g) At 48 hr post-infection with lentivirus containing scrambled shRNA (SC) or TRIM9-specific shRNA (KD), 293T cells expressing V5-TRIM9 were used for IP with anti-V5, followed by IB with the indicated antibodies. The data are representative of three independent experiments.
Mentions: To test whether serine phosphorylation of the TRIM9 degron motif is required for its interaction with β-TrCP, immunopurified HA-TRIM9 complexes were treated with mock or λ-phosphatase, incubated with lysates containing Flag-β-TrCP, and then subjected to immunoblotting with anti-Flag antibody. This showed that treatment of immunopurified TRIM9 with λ-phosphatase drastically abrogated its β-TrCP-binding ability (Fig. 3a). Phosphoserine (Sep), the most abundant phosphoamino acid in the eukaryotic phosphoproteome, is not encoded in the genetic code, but is synthesized post translationally. Since our attempts to generate the pS76pS80 phospho-specific TRIM9 antibodies failed twice, we utilized the specific cotranslational Sep incorporation system38, 39to study the Ser76 residue of TRIM9. An Escherichia coli strain was genetically engineered to harbor a Sep-accepting transfer RNA (tRNASep), its cognate Sep–tRNAsynthetase (SepRS), and an engineered EF-Tu (EF-Sep) of Methanocaldococcus jannaschii, and then transformed with bacterial expression vector containing the N-terminal HA-tagged RING domain (1-160aa) of TRIM9 WT, the SA mutant, and the non-natural Sep-incorporated pS76(directed by UAG) mutant (Fig. 3b). These HA-TRIM9 proteins were purified from genetically engineered E. coli and used for in vitro pulldown (PD) assay with Flag-β-TrCP. This showed that the pS76 TRIM9 protein specifically and efficiently bound β-TrCP in vitro, whereas the WT and SA TRIM9 proteins did not bind (Fig. 3c, d), suggesting that the serine phosphorylation of the TRIM9 degron motif is necessary for binding the WD40 domain of β-TrCP.

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