Importance of the Conserved Carboxyl-Terminal CNOT1 Binding Domain to Tristetraprolin Activity <i>In Vivo</i>

Lai, Wi S.; Stumpo, Deborah J.; Wells, Melissa L.; Gruzdev, Artiom; Hicks, Stephanie; Nicholson, Cindo O.; Yang, Zhengfeng; Faccio, Roberta; Webster, M.; Passmore, Lori A.; Blackshear, Perry J.

doi:10.1128/mcb.00029-19

Cited by 17 publications

(21 citation statements)

References 54 publications

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“…There are four TTP members in rodents, including TTP, Zfp36l1, Zfp36l2, and Zfp36l3, and they all contain conserved tandem CCCH zinc finger RNA-binding domains and a conserved C-terminal NOT1-binding domain [14]. TTP contains intrinsically unstructured regions outside these two conserved regions [15][16][17]. The metazoa type of NOT1-binding domain is missing in most fungi; for examples, the TTP homologs CTH1 and CTH2 in Saccharomyces cerevisiae, and Zfs1p in S. pombe, are no containing NOT1-binding domain [14].…”

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confidence: 99%

The functional characterization of phosphorylation of tristetraprolin at C-terminal NOT1-binding domain

Hsieh

Chen

Chang³

et al. 2021

J Inflamm

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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.

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confidence: 99%

The functional characterization of phosphorylation of tristetraprolin at C-terminal NOT1-binding domain

Hsieh

Chen

Chang³

et al. 2021

J Inflamm

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“…Studies examining the structural basis of TTP's association with the central cytoplasmic deadenylase, the CCR4-NOT complex, revealed an interaction with the CCR4-NOT scaffold protein, CNOT1, and this C-terminal region of TTP, which was therefore named the CNOT1-Interacting Motif (CIM) (8,22). Supporting the importance of the CIM, deletion of the TTP CIM led to a stabilization of target transcripts (8,23).…”

Section: Introductionmentioning

confidence: 99%

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

Carreno

Lykke-Andersen

2022

Preprint

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Regulation of the mRNA decay activator Tristetraprolin (TTP) by the p38 mitogen-activated protein kinase (MAPK) pathway during the mammalian inflammatory response represents a paradigm for the regulation of mRNA turnover by signaling. Phosphorylation of TTP by p38 MAPK-activated kinase 2 (MK2) inhibits the association of TTP with the CCR4-NOT deadenylase complex and represses TTP-mediated mRNA decay. Here we present evidence that TTP remains active in the presence of activated MK2 due to its highly conserved CNOT1 Interacting Motif (CIM), which remains unphosphorylated and capable of promoting deadenylation and decay. The CIM recruits the CCR4-NOT complex cooperatively with previously identified conserved tryptophan residues of TTP and deletion of the CIM strongly represses residual association with the deadenylase complex and activity of TTP in conditions of active MK2. A conserved serine in the CIM is not a target of MK2 but is instead phosphorylated by other kinases including the PKCa pathway and regulates TTP activity independently of MK2. These results suggest that kinase pathways regulate TTP activity in a cooperative manner and that the p38 MAPK-MK2 pathway relies on the activation of additional kinase pathway(s) to fully control TTP function.

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“…Because NOT1 acts as an essential scaffold and yet lacks enzymatic activity of its own, recruitment of the appropriate effector proteins and their regulators is critical for the function of this complex. In addition to the proteins noted above, embryonic lethal abnormal vision (ELAV) family proteins and tristetraprolin (TTP) are recruited and act as antagonistic, mutually exclusive modulators to functionally toggle the complex’s activity between transcript preservation versus transcript degradation [ 27 ]. ELAV family proteins (which include HuR and CELF proteins) and TTPs (which are C3H1 zinc finger proteins and include ZFP36, TIS11) are RNA-binding proteins that bind with AU-rich elements in eukaryotes [ 28 ].…”

Section: Introductionmentioning

confidence: 99%

The Plasmodium NOT1-G paralogue is an essential regulator of sexual stage maturation and parasite transmission

et al. 2021

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Productive transmission of malaria parasites hinges upon the execution of key transcriptional and posttranscriptional regulatory events. While much is now known about how specific transcription factors activate or repress sexual commitment programs, far less is known about the production of a preferred mRNA homeostasis following commitment and through the host-to-vector transmission event. Here, we show that in Plasmodium parasites, the NOT1 scaffold protein of the CAF1/CCR4/Not complex is duplicated, and one paralogue is dedicated for essential transmission functions. Moreover, this NOT1-G paralogue is central to the sex-specific functions previously associated with its interacting partners, as deletion of not1-g in Plasmodium yoelii leads to a comparable or complete arrest phenotype for both male and female parasites. We show that, consistent with its role in other eukaryotes, PyNOT1-G localizes to cytosolic puncta throughout much of the Plasmodium life cycle. PyNOT1-G is essential to both the complete maturation of male gametes and to the continued development of the fertilized zygote originating from female parasites. Comparative transcriptomics of wild-type and pynot1-g− parasites shows that loss of PyNOT1-G leads to transcript dysregulation preceding and during gametocytogenesis and shows that PyNOT1-G acts to preserve mRNAs that are critical to sexual and early mosquito stage development. Finally, we demonstrate that the tristetraprolin (TTP)-binding domain, which acts as the typical organization platform for RNA decay (TTP) and RNA preservation (ELAV/HuR) factors is dispensable for PyNOT1-G’s essential blood stage functions but impacts host-to-vector transmission. Together, we conclude that a NOT1-G paralogue in Plasmodium fulfills the complex transmission requirements of both male and female parasites.

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Importance of the Conserved Carboxyl-Terminal CNOT1 Binding Domain to Tristetraprolin Activity In Vivo

Cited by 17 publications

References 54 publications

The functional characterization of phosphorylation of tristetraprolin at C-terminal NOT1-binding domain

The functional characterization of phosphorylation of tristetraprolin at C-terminal NOT1-binding domain

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

The Plasmodium NOT1-G paralogue is an essential regulator of sexual stage maturation and parasite transmission

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