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Polyubiquitination of Transforming Growth Factor β-activated Kinase 1 (TAK1) at Lysine 562 Residue Regulates TLR4-mediated JNK and p38 MAPK Activation.

Chen IT, Hsu PH, Hsu WC, Chen NJ, Tseng PH - Sci Rep (2015)

Bottom Line: Moreover, using LC-MS analysis, Lys562 of TAK1 was identified as a novel Lys63-linked ubiquitination site and as the key residue in the feedback regulation.Mutation of Lys562 of TAK1 leads to a decrease in TAK1 phosphorylation and specific inhibition of the MAPK pathway, but has no effect on formation of the TAK1-containing complex.Our findings demonstrate a feedback loop for phosphorylation and ubiquitination of TAK1, indicating a dynamic regulation between TAK1 polyubiquitiantion and phosphorylated activation, and the molecular mechanism by which IKK and MAPKs are differentially activated in the TLR4 pathway.

View Article: PubMed Central - PubMed

Affiliation: Institute of Biochemistry and Molecular Biology, School of Life Sciences, National Yang-Ming University, Taipei 11221, Taiwan (ROC).

ABSTRACT
Toll-like receptor 4 (TLR4) plays an important role in innate immunity by eliciting inflammation. Upon receptor engagement, transforming growth factor β-activated kinase 1 (TAK1) is an essential mediator that transmits a signal from the receptor to downstream effectors, IκB kinase (IKK) and the mitogen-activated protein kinases (MAPKs), which control the production of inflammatory cytokines. However, the association between phosphorylation and ubiquitination of TAK1 is not yet clear. Here, we examined the crosstalk between phosphorylation and polyubiquitination of TAK1 and further investigated the mechanism of distinct activation of MAPKs and IKK. Inhibition of TAK1 phosphorylation enhanced Lys63-linked polyubiquitination of TAK1. Conversely, ubiquitin modification was counteracted by phospho-mimic TAK1 mutant, T(184,187)D. Moreover, using LC-MS analysis, Lys562 of TAK1 was identified as a novel Lys63-linked ubiquitination site and as the key residue in the feedback regulation. Mutation of Lys562 of TAK1 leads to a decrease in TAK1 phosphorylation and specific inhibition of the MAPK pathway, but has no effect on formation of the TAK1-containing complex. Our findings demonstrate a feedback loop for phosphorylation and ubiquitination of TAK1, indicating a dynamic regulation between TAK1 polyubiquitiantion and phosphorylated activation, and the molecular mechanism by which IKK and MAPKs are differentially activated in the TLR4 pathway.

No MeSH data available.


Related in: MedlinePlus

Inhibition of polyubiquitination of TAK1 at Lys562 residue leads to a decline in cytokine production.A and B, TAK1 is required for TLR4-mediated induction of TNF and IL-6 mRNA. TAK1-silenced RAW264.7 cells were reconstituted with Flag-tagged TAK1 wild-type (WT), T(184,187)A, K562R or K(562,563)R mutant. After treatment with LPS for indicated times, the cells were collected and the total RNA was extracted, reverse transcribed, and analyzed for TNF and IL-6 mRNA with Q-PCR. C and D, TAK1 is required for TLR4-mediated TNF and IL-6 production. TAK1-silencing RAW264.7 cells were reconstituted with Flag-tagged TAK1 wild-type (WT), T(184,187)A, K562R or K(562,563)R mutant. After incubating with LPS for 24 h, the medium was collected and analyzed for TNF and IL-6 level with ELISA. Results are averages ± SD of three separate experiments (*, significant difference).
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f7: Inhibition of polyubiquitination of TAK1 at Lys562 residue leads to a decline in cytokine production.A and B, TAK1 is required for TLR4-mediated induction of TNF and IL-6 mRNA. TAK1-silenced RAW264.7 cells were reconstituted with Flag-tagged TAK1 wild-type (WT), T(184,187)A, K562R or K(562,563)R mutant. After treatment with LPS for indicated times, the cells were collected and the total RNA was extracted, reverse transcribed, and analyzed for TNF and IL-6 mRNA with Q-PCR. C and D, TAK1 is required for TLR4-mediated TNF and IL-6 production. TAK1-silencing RAW264.7 cells were reconstituted with Flag-tagged TAK1 wild-type (WT), T(184,187)A, K562R or K(562,563)R mutant. After incubating with LPS for 24 h, the medium was collected and analyzed for TNF and IL-6 level with ELISA. Results are averages ± SD of three separate experiments (*, significant difference).

Mentions: TAK1 mediates IKK and MAPK activation which in turn induce inflammatory cytokines, such as TNF and IL-6. Next, the role of Lys562 in TAK1-mediated gene expression was investigated. TAK1-silenced RAW264.7 cells were reconstituted with either TAK1 WT, T(184,187)A, K562R, or K(562,563)R mutants, and TAK1-induced mRNA expression, including that of TNF and IL6, in response to LPS stimulation was analyzed with RT-qPCR (Fig. 7A,B). It was shown previously that Lys562 residue of TAK1 is critical for MAPKs activation. Meanwhile, MAPKs pathway has been indicated to regulate inflammatory cytokines, including TNF and IL-6 through multiple mechanisms373839. Therefore, it was no surprise that the mRNA level of TNF and IL-6 was decreased in cells expressing K562R or K(562,563)R mutants. In TAK1-silenced THP-1 cells, TNF and IL-6 mRNA production in response to LPS was also decreased with reconstitution of K(562,563)R mutant (Supplementary Figure 8A and B). Besides, T(184,187)A mutant that inhibits MAPK signaling also caused inhibition in TNF and IL-6 mRNA production (Fig. 7A,B). To determinate the protein level of cytokines, TAK1-knock down RAW264.7 cells were reconstituted with TAK1 WT, T(184,187)A, K562R, or K(562,563)R mutants. The production of cytokines, such as TNF and IL-6, was detected by ELISA (Fig. 7C,D). In line with the finding that mutation in Lys562 site of TAK1 had a decreased level of mRNA expression of inflammatory cytokines, K562R or K(562,563)R mutant led to the decline of TNF and IL-6 production in response to TLR4 signaling. Furthermore, in line with previous result that Lys562 residue was also critical in TLR2-mediated MAPK activation (Supplementary Figure 6D), the decreased IL-6 mRNA expression could be observed in TAK1-knocked down RAW264.7 cells reconstituted with T(184,187)A or K(562,563)R TAK1 mutants in response to Pam3CSK4 stimulation (Supplementary Figure 8C).


Polyubiquitination of Transforming Growth Factor β-activated Kinase 1 (TAK1) at Lysine 562 Residue Regulates TLR4-mediated JNK and p38 MAPK Activation.

Chen IT, Hsu PH, Hsu WC, Chen NJ, Tseng PH - Sci Rep (2015)

Inhibition of polyubiquitination of TAK1 at Lys562 residue leads to a decline in cytokine production.A and B, TAK1 is required for TLR4-mediated induction of TNF and IL-6 mRNA. TAK1-silenced RAW264.7 cells were reconstituted with Flag-tagged TAK1 wild-type (WT), T(184,187)A, K562R or K(562,563)R mutant. After treatment with LPS for indicated times, the cells were collected and the total RNA was extracted, reverse transcribed, and analyzed for TNF and IL-6 mRNA with Q-PCR. C and D, TAK1 is required for TLR4-mediated TNF and IL-6 production. TAK1-silencing RAW264.7 cells were reconstituted with Flag-tagged TAK1 wild-type (WT), T(184,187)A, K562R or K(562,563)R mutant. After incubating with LPS for 24 h, the medium was collected and analyzed for TNF and IL-6 level with ELISA. Results are averages ± SD of three separate experiments (*, significant difference).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4507259&req=5

f7: Inhibition of polyubiquitination of TAK1 at Lys562 residue leads to a decline in cytokine production.A and B, TAK1 is required for TLR4-mediated induction of TNF and IL-6 mRNA. TAK1-silenced RAW264.7 cells were reconstituted with Flag-tagged TAK1 wild-type (WT), T(184,187)A, K562R or K(562,563)R mutant. After treatment with LPS for indicated times, the cells were collected and the total RNA was extracted, reverse transcribed, and analyzed for TNF and IL-6 mRNA with Q-PCR. C and D, TAK1 is required for TLR4-mediated TNF and IL-6 production. TAK1-silencing RAW264.7 cells were reconstituted with Flag-tagged TAK1 wild-type (WT), T(184,187)A, K562R or K(562,563)R mutant. After incubating with LPS for 24 h, the medium was collected and analyzed for TNF and IL-6 level with ELISA. Results are averages ± SD of three separate experiments (*, significant difference).
Mentions: TAK1 mediates IKK and MAPK activation which in turn induce inflammatory cytokines, such as TNF and IL-6. Next, the role of Lys562 in TAK1-mediated gene expression was investigated. TAK1-silenced RAW264.7 cells were reconstituted with either TAK1 WT, T(184,187)A, K562R, or K(562,563)R mutants, and TAK1-induced mRNA expression, including that of TNF and IL6, in response to LPS stimulation was analyzed with RT-qPCR (Fig. 7A,B). It was shown previously that Lys562 residue of TAK1 is critical for MAPKs activation. Meanwhile, MAPKs pathway has been indicated to regulate inflammatory cytokines, including TNF and IL-6 through multiple mechanisms373839. Therefore, it was no surprise that the mRNA level of TNF and IL-6 was decreased in cells expressing K562R or K(562,563)R mutants. In TAK1-silenced THP-1 cells, TNF and IL-6 mRNA production in response to LPS was also decreased with reconstitution of K(562,563)R mutant (Supplementary Figure 8A and B). Besides, T(184,187)A mutant that inhibits MAPK signaling also caused inhibition in TNF and IL-6 mRNA production (Fig. 7A,B). To determinate the protein level of cytokines, TAK1-knock down RAW264.7 cells were reconstituted with TAK1 WT, T(184,187)A, K562R, or K(562,563)R mutants. The production of cytokines, such as TNF and IL-6, was detected by ELISA (Fig. 7C,D). In line with the finding that mutation in Lys562 site of TAK1 had a decreased level of mRNA expression of inflammatory cytokines, K562R or K(562,563)R mutant led to the decline of TNF and IL-6 production in response to TLR4 signaling. Furthermore, in line with previous result that Lys562 residue was also critical in TLR2-mediated MAPK activation (Supplementary Figure 6D), the decreased IL-6 mRNA expression could be observed in TAK1-knocked down RAW264.7 cells reconstituted with T(184,187)A or K(562,563)R TAK1 mutants in response to Pam3CSK4 stimulation (Supplementary Figure 8C).

Bottom Line: Moreover, using LC-MS analysis, Lys562 of TAK1 was identified as a novel Lys63-linked ubiquitination site and as the key residue in the feedback regulation.Mutation of Lys562 of TAK1 leads to a decrease in TAK1 phosphorylation and specific inhibition of the MAPK pathway, but has no effect on formation of the TAK1-containing complex.Our findings demonstrate a feedback loop for phosphorylation and ubiquitination of TAK1, indicating a dynamic regulation between TAK1 polyubiquitiantion and phosphorylated activation, and the molecular mechanism by which IKK and MAPKs are differentially activated in the TLR4 pathway.

View Article: PubMed Central - PubMed

Affiliation: Institute of Biochemistry and Molecular Biology, School of Life Sciences, National Yang-Ming University, Taipei 11221, Taiwan (ROC).

ABSTRACT
Toll-like receptor 4 (TLR4) plays an important role in innate immunity by eliciting inflammation. Upon receptor engagement, transforming growth factor β-activated kinase 1 (TAK1) is an essential mediator that transmits a signal from the receptor to downstream effectors, IκB kinase (IKK) and the mitogen-activated protein kinases (MAPKs), which control the production of inflammatory cytokines. However, the association between phosphorylation and ubiquitination of TAK1 is not yet clear. Here, we examined the crosstalk between phosphorylation and polyubiquitination of TAK1 and further investigated the mechanism of distinct activation of MAPKs and IKK. Inhibition of TAK1 phosphorylation enhanced Lys63-linked polyubiquitination of TAK1. Conversely, ubiquitin modification was counteracted by phospho-mimic TAK1 mutant, T(184,187)D. Moreover, using LC-MS analysis, Lys562 of TAK1 was identified as a novel Lys63-linked ubiquitination site and as the key residue in the feedback regulation. Mutation of Lys562 of TAK1 leads to a decrease in TAK1 phosphorylation and specific inhibition of the MAPK pathway, but has no effect on formation of the TAK1-containing complex. Our findings demonstrate a feedback loop for phosphorylation and ubiquitination of TAK1, indicating a dynamic regulation between TAK1 polyubiquitiantion and phosphorylated activation, and the molecular mechanism by which IKK and MAPKs are differentially activated in the TLR4 pathway.

No MeSH data available.


Related in: MedlinePlus