Hsin-Hui Hsieh1, Yen-An Chen1, Yao-Jen Chang2, Hsin-Hui Wang3,4,5, Ya-Han Yu1, Sheng-Wei Lin2, Yin-Jung Huang3, Steven Lin1,2, Ching-Jin Chang6,7. 1. Graduate Institute of Biochemical Sciences, College of Life Science, National Taiwan University, No. 1 Sec 4 Roosevelt Rd, Taipei, 106, Taiwan. 2. Institute of Biological Chemistry, Academia Sinica, Taipei, Taiwan. 3. Department of Pediatrics, Division of Pediatric Immunology and Nephrology, Taipei Veterans General Hospital, Taipei, Taiwan. 4. Department of Pediatrics, Faculty of Medicine, School of Medicine, National Yang Ming Chiao Tung University, Taipei, Taiwan. 5. Institute of Emergency and Critical Care Medicine, School of Medicine, National Yang Ming Chiao Tung University, Taipei, Taiwan. 6. Graduate Institute of Biochemical Sciences, College of Life Science, National Taiwan University, No. 1 Sec 4 Roosevelt Rd, Taipei, 106, Taiwan. chingjin@gate.sinica.edu.tw. 7. Institute of Biological Chemistry, Academia Sinica, Taipei, Taiwan. chingjin@gate.sinica.edu.tw.
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.
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 TTPphosphorylation 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 S316ATTP mutant can restore TTP activity and lead to TNFα mRNA decreased. GST pull-down and RNA pull-down analyses demonstrated that endogenous TTP with Ser316phosphorylation decreased the interaction with CNOT1. CONCLUSIONS: Our results suggest that the TTP-mediated mRNA stability is modulated by Ser316phosphorylation via regulating the TTP interaction with the CCR4-NOT deadenylase complex.
Authors: Laia Castells-Roca; José García-Martínez; Joaquín Moreno; Enrique Herrero; Gemma Bellí; José E Pérez-Ortín Journal: PLoS One Date: 2011-02-25 Impact factor: 3.240
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