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 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.

The Conserved CNOT1 Interaction Motif of Tristetraprolin Regulates ARE-mRNA Decay Independently of the p38 MAPK-MK2 Kinase Pathway

“…4A ). Consistent with a recent report ( 29 ), induction of the inflammatory response by serum shock of mouse NIH3T3 fibroblasts ( Fig. 4B ), or treatment with lipopolysaccharide (LPS) of mouse RAW264.7 macrophages ( Fig.…”

“…The TTP-CIM has been reported as a target of several kinase pathways ( 10 , 29 – 31 ), including the p38 MAPK-MK2 pathway, which also targets TTP serines 52 and 178 ( 10 , 28 ). HeLa cells transiently expressing TTP show detectable levels of phosphorylation of both serines 178 and 316 of TTP ( Fig.…”

“…Another serine of TTP that undergoes phosphorylation is serine 316 (mouse numbering), an evolutionary conserved serine of the CIM motif. Phosphorylation of this residue has been shown to inhibit association of TTP-family proteins with CNOT1, suggesting that the association of the major deadenylase complex is repressed by phosphorylation ( 8 , 29 , 30 ). However, a separate study using phosphomimetic mutations, suggested that phosphorylation of the CIM of the TTP homolog BRF1 promotes its association with decapping factors and accelerates mRNA decay ( 30 ).…”

“…However, a separate study using phosphomimetic mutations, suggested that phosphorylation of the CIM of the TTP homolog BRF1 promotes its association with decapping factors and accelerates mRNA decay ( 30 ). Although several studies have identified the CIM as a target of MK2 ( 10 , 29 , 31 ), others have implicated the kinases RSK1 and PKA ( 29 , 30 ). Thus, the importance of the highly conserved CIM and its phosphorylation in TTP regulation and function remains poorly defined.…”

The Emerging Role of Stress Granules in Hepatocellular Carcinoma

“…It contains a zinc finger domain with a double zinc finger motif (Cys-Cys-Cys-His) responsible for RNA binding, three quadruple proline motifs responsible for the binding of the 4EHP (a.k.a. eIF4E2) -GYF2 (GRB10-interacting GYF protein 2) cap-binding complex, and a Not-1 binding domain [120][121][122]. It is usually described as a tumor suppressor, downregulated in many human cancers, and its level correlates with a poor clinical outcome [123][124][125].…”