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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

Lys562 residue of TAK1 is an ubiquitination site.A, Coomassie blue stained gel indicates the band with TAK1 polyubiquitination. Flag-tagged TAK1 with or without HA-tagged ubiquitin were expressed in HEK293T cells. Cells were lysed, immunopricipitated, gel-separated, and stained with Coomassie blue. The square area indicated the presence of ubiquinated-TAK1. B, Lys562 residue of TAK1 was identified as an ubiquitination site by mass spectrometry. The protein sample with ubiquitinated TAK1 was recovered from the gel, enzyme digested, and subjected to mass analysis. Representative MS/MS spectra of peptides demonstrated ubiquitination at Lys562 of TAK1. Peaks matching expected b and y ions are labeled. C, Upper panel: schematic structure of TAK1, indicating functional domains and the new site identified in this study. Lower panel: alignment of part of the C-terminal of TAK1. The Lys562 residue is indicated by an asterisk.
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f3: Lys562 residue of TAK1 is an ubiquitination site.A, Coomassie blue stained gel indicates the band with TAK1 polyubiquitination. Flag-tagged TAK1 with or without HA-tagged ubiquitin were expressed in HEK293T cells. Cells were lysed, immunopricipitated, gel-separated, and stained with Coomassie blue. The square area indicated the presence of ubiquinated-TAK1. B, Lys562 residue of TAK1 was identified as an ubiquitination site by mass spectrometry. The protein sample with ubiquitinated TAK1 was recovered from the gel, enzyme digested, and subjected to mass analysis. Representative MS/MS spectra of peptides demonstrated ubiquitination at Lys562 of TAK1. Peaks matching expected b and y ions are labeled. C, Upper panel: schematic structure of TAK1, indicating functional domains and the new site identified in this study. Lower panel: alignment of part of the C-terminal of TAK1. The Lys562 residue is indicated by an asterisk.

Mentions: Since the enhanced polyubiquitination caused by inhibition of TAK1 activation was not at Lys158, it is very likely that TAK1 contains multiple ubiquitination sites in response to signal activation. Flag-tagged TAK1 and ubiquitin were co-expressed in HEK293T cells to force ubiquitination. After immunoprecipitating ubiquitinated TAK1 with anti-Flag antibody, the sample was separated by SDS-PAGE and visualized by Coomassie blue (Fig. 3A). In order to analyze ubiquitinated TAK1 by MS, the gel region containing ubiquitin-modified protein was excised, digested with trypsin, and analyzed by LC-MS/MS. A database search of the MS/MS spectra revealed that TAK1 was the predominant protein identified in the samples. Representative spectra that demonstrate that the Lys562 residue of TAK1 was identified to be modified with ubiquitin with 60% sequence coverage are shown in Fig. 3B. Comparison of the C-terminal protein sequence of TAK1 in different species showed that the Lys562 residue was highly conserved (Fig. 3C).


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)

Lys562 residue of TAK1 is an ubiquitination site.A, Coomassie blue stained gel indicates the band with TAK1 polyubiquitination. Flag-tagged TAK1 with or without HA-tagged ubiquitin were expressed in HEK293T cells. Cells were lysed, immunopricipitated, gel-separated, and stained with Coomassie blue. The square area indicated the presence of ubiquinated-TAK1. B, Lys562 residue of TAK1 was identified as an ubiquitination site by mass spectrometry. The protein sample with ubiquitinated TAK1 was recovered from the gel, enzyme digested, and subjected to mass analysis. Representative MS/MS spectra of peptides demonstrated ubiquitination at Lys562 of TAK1. Peaks matching expected b and y ions are labeled. C, Upper panel: schematic structure of TAK1, indicating functional domains and the new site identified in this study. Lower panel: alignment of part of the C-terminal of TAK1. The Lys562 residue is indicated by an asterisk.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f3: Lys562 residue of TAK1 is an ubiquitination site.A, Coomassie blue stained gel indicates the band with TAK1 polyubiquitination. Flag-tagged TAK1 with or without HA-tagged ubiquitin were expressed in HEK293T cells. Cells were lysed, immunopricipitated, gel-separated, and stained with Coomassie blue. The square area indicated the presence of ubiquinated-TAK1. B, Lys562 residue of TAK1 was identified as an ubiquitination site by mass spectrometry. The protein sample with ubiquitinated TAK1 was recovered from the gel, enzyme digested, and subjected to mass analysis. Representative MS/MS spectra of peptides demonstrated ubiquitination at Lys562 of TAK1. Peaks matching expected b and y ions are labeled. C, Upper panel: schematic structure of TAK1, indicating functional domains and the new site identified in this study. Lower panel: alignment of part of the C-terminal of TAK1. The Lys562 residue is indicated by an asterisk.
Mentions: Since the enhanced polyubiquitination caused by inhibition of TAK1 activation was not at Lys158, it is very likely that TAK1 contains multiple ubiquitination sites in response to signal activation. Flag-tagged TAK1 and ubiquitin were co-expressed in HEK293T cells to force ubiquitination. After immunoprecipitating ubiquitinated TAK1 with anti-Flag antibody, the sample was separated by SDS-PAGE and visualized by Coomassie blue (Fig. 3A). In order to analyze ubiquitinated TAK1 by MS, the gel region containing ubiquitin-modified protein was excised, digested with trypsin, and analyzed by LC-MS/MS. A database search of the MS/MS spectra revealed that TAK1 was the predominant protein identified in the samples. Representative spectra that demonstrate that the Lys562 residue of TAK1 was identified to be modified with ubiquitin with 60% sequence coverage are shown in Fig. 3B. Comparison of the C-terminal protein sequence of TAK1 in different species showed that the Lys562 residue was highly conserved (Fig. 3C).

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