The functional characterization of phosphorylation of tristetraprolin at C-terminal NOT1-binding domain
Abstract Background Tristetraprolin (TTP) family proteins contain conserved tandem CCCH zinc-finger binding to AU-rich elements and C-terminal NOT1-binding domain. TTP is phosphorylated extensively in cells, and its mRNA destabilization activity is regulated by protein phosphorylation. Methods We ge...
| Main Authors: | , , , , , , , , |
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| Format: | Article |
| Language: | English |
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BMC
2021-06-01
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| Series: | Journal of Inflammation |
| Subjects: | |
| Online Access: | https://doi.org/10.1186/s12950-021-00288-2 |
| _version_ | 1818666752930742272 |
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| author | Hsin-Hui Hsieh Yen-An Chen Yao-Jen Chang Hsin-Hui Wang Ya-Han Yu Sheng-Wei Lin Yin-Jung Huang Steven Lin Ching-Jin Chang |
| author_facet | Hsin-Hui Hsieh Yen-An Chen Yao-Jen Chang Hsin-Hui Wang Ya-Han Yu Sheng-Wei Lin Yin-Jung Huang Steven Lin Ching-Jin Chang |
| author_sort | Hsin-Hui Hsieh |
| collection | DOAJ |
| description | Abstract Background Tristetraprolin (TTP) family proteins contain conserved tandem CCCH zinc-finger binding to AU-rich elements and C-terminal NOT1-binding domain. TTP is phosphorylated extensively in cells, and its mRNA destabilization activity is regulated by protein phosphorylation. Methods We generated an antibody against phospho-Serine316 located at the C-terminal NOT1-binding site and examined TTP phosphorylation in LPS-stimulated RAW264.7 cells. Knockout of TTP was created in RAW264.7 cells using CRISPR/Cas9 gene editing to explore TTP functions. Results We demonstrated that Ser316 was phosphorylated by p90 ribosomal S6 kinase 1 (RSK1) and p38-activated protein kinase (MK2) and dephosphorylated by Protein Phosphatase 2A (PP2A). A phosphorylation-mimic mutant of S316D resulted in dissociation with the CCR4-NOT deadenylase complex through weakening interaction with CNOT1. Furthermore, Ser316 and serines 52 and 178 were independently contributed to the CCR4-NOT complex recruitment in the immunoprecipitation assay using phosphor-mimic mutants. In RAW264.7 macrophages, TTP was induced, and Ser316 was phosphorylated through RSK1 and MK2 by LPS stimulation. Knockout of TTP resulted in TNFα mRNA increased due to mRNA stabilization. Overexpression of non-phosphorylated S316A TTP mutant can restore TTP activity and lead to TNFα mRNA decreased. GST pull-down and RNA pull-down analyses demonstrated that endogenous TTP with Ser316 phosphorylation decreased the interaction with CNOT1. Conclusions Our results suggest that the TTP-mediated mRNA stability is modulated by Ser316 phosphorylation via regulating the TTP interaction with the CCR4-NOT deadenylase complex. |
| first_indexed | 2024-12-17T06:09:32Z |
| format | Article |
| id | doaj.art-56eec6847e0b49c48b9788359b12a257 |
| institution | Directory Open Access Journal |
| issn | 1476-9255 |
| language | English |
| last_indexed | 2024-12-17T06:09:32Z |
| publishDate | 2021-06-01 |
| publisher | BMC |
| record_format | Article |
| series | Journal of Inflammation |
| spelling | doaj.art-56eec6847e0b49c48b9788359b12a2572022-12-21T22:00:40ZengBMCJournal of Inflammation1476-92552021-06-0118111610.1186/s12950-021-00288-2The functional characterization of phosphorylation of tristetraprolin at C-terminal NOT1-binding domainHsin-Hui Hsieh0Yen-An Chen1Yao-Jen Chang2Hsin-Hui Wang3Ya-Han Yu4Sheng-Wei Lin5Yin-Jung Huang6Steven Lin7Ching-Jin Chang8Graduate Institute of Biochemical Sciences, College of Life Science, National Taiwan UniversityGraduate Institute of Biochemical Sciences, College of Life Science, National Taiwan UniversityInstitute of Biological ChemistryDepartment of Pediatrics, Division of Pediatric Immunology and Nephrology, Taipei Veterans General HospitalGraduate Institute of Biochemical Sciences, College of Life Science, National Taiwan UniversityInstitute of Biological ChemistryDepartment of Pediatrics, Division of Pediatric Immunology and Nephrology, Taipei Veterans General HospitalGraduate Institute of Biochemical Sciences, College of Life Science, National Taiwan UniversityGraduate Institute of Biochemical Sciences, College of Life Science, National Taiwan UniversityAbstract Background Tristetraprolin (TTP) family proteins contain conserved tandem CCCH zinc-finger binding to AU-rich elements and C-terminal NOT1-binding domain. TTP is phosphorylated extensively in cells, and its mRNA destabilization activity is regulated by protein phosphorylation. Methods We generated an antibody against phospho-Serine316 located at the C-terminal NOT1-binding site and examined TTP phosphorylation in LPS-stimulated RAW264.7 cells. Knockout of TTP was created in RAW264.7 cells using CRISPR/Cas9 gene editing to explore TTP functions. Results We demonstrated that Ser316 was phosphorylated by p90 ribosomal S6 kinase 1 (RSK1) and p38-activated protein kinase (MK2) and dephosphorylated by Protein Phosphatase 2A (PP2A). A phosphorylation-mimic mutant of S316D resulted in dissociation with the CCR4-NOT deadenylase complex through weakening interaction with CNOT1. Furthermore, Ser316 and serines 52 and 178 were independently contributed to the CCR4-NOT complex recruitment in the immunoprecipitation assay using phosphor-mimic mutants. In RAW264.7 macrophages, TTP was induced, and Ser316 was phosphorylated through RSK1 and MK2 by LPS stimulation. Knockout of TTP resulted in TNFα mRNA increased due to mRNA stabilization. Overexpression of non-phosphorylated S316A TTP mutant can restore TTP activity and lead to TNFα mRNA decreased. GST pull-down and RNA pull-down analyses demonstrated that endogenous TTP with Ser316 phosphorylation decreased the interaction with CNOT1. Conclusions Our results suggest that the TTP-mediated mRNA stability is modulated by Ser316 phosphorylation via regulating the TTP interaction with the CCR4-NOT deadenylase complex.https://doi.org/10.1186/s12950-021-00288-2TristetraprolinPhosphorylationCNOT1, deadenylase |
| spellingShingle | Hsin-Hui Hsieh Yen-An Chen Yao-Jen Chang Hsin-Hui Wang Ya-Han Yu Sheng-Wei Lin Yin-Jung Huang Steven Lin Ching-Jin Chang The functional characterization of phosphorylation of tristetraprolin at C-terminal NOT1-binding domain Journal of Inflammation Tristetraprolin Phosphorylation CNOT1, deadenylase |
| title | The functional characterization of phosphorylation of tristetraprolin at C-terminal NOT1-binding domain |
| title_full | The functional characterization of phosphorylation of tristetraprolin at C-terminal NOT1-binding domain |
| title_fullStr | The functional characterization of phosphorylation of tristetraprolin at C-terminal NOT1-binding domain |
| title_full_unstemmed | The functional characterization of phosphorylation of tristetraprolin at C-terminal NOT1-binding domain |
| title_short | The functional characterization of phosphorylation of tristetraprolin at C-terminal NOT1-binding domain |
| title_sort | functional characterization of phosphorylation of tristetraprolin at c terminal not1 binding domain |
| topic | Tristetraprolin Phosphorylation CNOT1, deadenylase |
| url | https://doi.org/10.1186/s12950-021-00288-2 |
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