Tristetraprolin (TTP)-14-3-3 Complex Formation Protects TTP from Dephosphorylation by Protein Phosphatase 2a and Stabilizes Tumor Necrosis Factor-α mRNA

Sun, Lei; Stoecklin, Georg; Way, Susan Van; Hinkovska‐Galcheva, Vania; Guo, Ruocheng; Anderson, Paul; Shanley, Thomas P.

doi:10.1074/jbc.m607347200

Cited by 176 publications

(194 citation statements)

References 52 publications

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“…Phosphorylation of TTP by MK2 has been suggested to reduce the affinity of TTP for ARE-containing mRNA (7). However, others found that mutation of the two major MK2 phosphorylation sites to alanine had no effect on ARE binding (42). Using purified recombinant TTP and MK2, we also could not detect any difference in affinity of unphosphorylated or phosphorylated TTP for the GM-CSF ARE, showing that regulation of RNA binding does not account for the regulation of deadenylation observed in the in vitro system described here.…”

Section: Discussioncontrasting

confidence: 55%

MAPKAP Kinase 2 Blocks Tristetraprolin-directed mRNA Decay by Inhibiting CAF1 Deadenylase Recruitment

Marchese

Aubareda

Tudor³

et al. 2010

Journal of Biological Chemistry

136

149

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Tristetraprolin (TTP) directs its target AU-rich element (ARE)-containing mRNAs for degradation by promoting removal of the poly(A) tail. The p38 MAPK pathway regulates mRNA stability via the downstream kinase MAPK-activated protein kinase 2 (MAPKAP kinase 2 or MK2), which phosphorylates and prevents the mRNA-destabilizing function of TTP. We show that deadenylation of endogenous ARE-containing tumor necrosis factor mRNA is inhibited by p38 MAPK. To investigate whether phosphorylation of TTP by MK2 regulates TTP-directed deadenylation of ARE-containing mRNAs, we used a cell-free assay that reconstitutes the mechanism in vitro. We find that phosphorylation of Ser-52 and Ser-178 of TTP by MK2 results in inhibition of TTP-directed deadenylation of ARE-containing RNA. The use of 14-3-3 protein antagonists showed that regulation of TTP-directed deadenylation by MK2 is independent of 14-3-3 binding to TTP. To investigate the mechanism whereby TTP promotes deadenylation, it was necessary to identify the deadenylases involved. The carbon catabolite repressor protein (CCR)4⅐CCR4-associated factor (CAF)1 complex was identified as the major source of deadenylase activity in HeLa cells responsible for TTP-directed deadenylation. CAF1a and CAF1b were found to interact with TTP in an RNAindependent fashion. We find that MK2 phosphorylation reduces the ability of TTP to promote deadenylation by inhibiting the recruitment of CAF1 deadenylase in a mechanism that does not involve sequestration of TTP by 14-3-3. Cyclooxygenase-2 mRNA stability is increased in CAF1-depleted cells in which it is no longer p38 MAPK/MK2-regulated.Many mammalian mRNAs contain adenosine/uridine-rich elements (AREs) 3 in their 3Ј-untranslated regions (UTRs) that target mRNAs for rapid degradation. The importance of AREs in regulation of mRNA stability has been demonstrated particularly for mRNAs of the inflammatory response. The p38 MAPK pathway inhibits ARE-mediated decay allowing for dynamic control of the expression of these mRNAs (1-3). p38 MAPK regulates mRNA stability via the downstream kinase MAPKAP kinase 2 (MK2), which phosphorylates (4, 5) and prevents the function (6, 7) of the mRNA-destabilizing ARE-binding protein, tristetraprolin (TTP). We recently showed that dual control of mRNA stability by TTP and the p38 MAPK pathway is a general mechanism, which operates for many mRNAs of the inflammatory response (8). The importance of TTP in the control of inflammatory gene expression is demonstrated by spontaneous inflammatory arthritis in TTP Ϫ/Ϫ mice arising mainly from increased tumor necrosis factor (TNF) production (9). p38 MAPK also regulates mRNA stability by direct phosphorylation and inactivation of another ARE-binding protein, KH domain-splicing regulatory protein (10, 11). p38 MAPK inhibitors fail to destabilize mRNAs of the inflammatory response in macrophages from TTP Ϫ/Ϫ mice (8, 12). Thus, it appears that in these cells, at least, TTP is entirely responsible for the effects on mRNA stability mediated by the p38 MAPK pathway.In additi...

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Section: Discussioncontrasting

confidence: 55%

MAPKAP Kinase 2 Blocks Tristetraprolin-directed mRNA Decay by Inhibiting CAF1 Deadenylase Recruitment

Marchese

Aubareda

Tudor³

et al. 2010

Journal of Biological Chemistry

136

149

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“…In addition, the regulation of both subcellular localization and protein stability of mTTP is dependent on MK2 and on the integrity of S 52 and S 178 [53]. Finally, mutation of S 52 to A 52 in mTTP weakly reduces the assembly of TTP-14-3-3, whereas mutation of S 178 to A 178 and of S 52/178 to A 52/178 substantially reduces the association of TTP with 14-3-3 [54].…”

Section: Introductionmentioning

confidence: 99%

Phosphorylation site analysis of the anti-inflammatory and mRNA-destabilizing protein tristetraprolin

Cao¹,

Deterding²,

Blackshear³

2007

Expert Review of Proteomics

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Tristetraprolin (TTP) is a member of the CCCH zinc finger proteins and is an anti-inflammatory protein. Mice deficient in TTP develop a profound inflammatory syndrome with erosive arthritis, autoimmunity and myeloid hyperplasia. TTP binds to mRNA AU-rich elements with high affinity for UUAUUUAUU nucleotides and causes destabilization of those mRNA molecules. TTP is phosphorylated extensively in vivo and is a substrate for multiple protein kinases in vitro. A number of approaches have been used to identify its phosphorylation sites. This article highlights the recent progress and different approaches utilized for the identification of phosphorylation sites in mammalian TTP. Important but limited results are obtained using traditional methods including in vivo labeling, site-directed mutagenesis, phosphopeptide mapping and protein sequencing. Mass spectrometry including MALDI/MS, MALDI/MS/MS, LC/MS/MS, IMAC/MALDI/MS/MS and multidimensional protein identification technology (MudPIT) has led the way in identifying TTP phosphorylation sites. The combination of these approaches has identified multiple phosphorylation sites in mammalian TTP, some of which are predicted by motif scanning to be phosphorylated by several protein kinases. This information should provide the molecular basis for future investigation of TTP's regulatory functions in controlling pro-inflammatory cytokines.

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“…[162][163][164] Eventually, the phosphatase PP2A binds to and dephosphorylates TTP, facilitating resumption of TTPmediated transcript decay. 158 A similar mechanism may link other kinases to downregulation of RNA destabilizing activity. Gherzi's laboratory has demonstrated that AKT phosphorylates KSRP on serine 193, creating a docking site for 14-3-3.…”

Section: Signaling Pathways and Mrna Turnover Controlmentioning

confidence: 99%

“…Transcript stability is controlled by kinases and other mediators characteristic of cytokine signal transduction pathways, including PKC, [146][147][148][149][150] PKB, 151 MAPK/Erk, 45 PI-3-kinase, 128,152 Src-family kinases, 153 p38 MAPK (reviewed in Dean et al 154 and Kotlyarov and Gaestel 155 ), mixed lineage kinase, 130 JNK kinases, 156,157 and phosphatases such as calcineurin 127 and PP2A. 158 Several of these signaling intermediaries, particularly p38 and ERK have been shown to be important in stabilizing inflammatory transcripts downstream of LPS or IL-1. The example below relates how phosphorylation cascades can lead to stabilization of TNFa in myeloid cells.…”

Section: Signaling Pathways and Mrna Turnover Controlmentioning

confidence: 99%

“…The destabilizing activity of TTP is coincidently suppressed. The mechanism for this has been controversial, 162 with some 163 but not all 158 studies showing that MK2 phosphorylated TTP had decreased affinity for target AREs. The association of 14-3-3 appears to prevent recruitment of TTP complexes to the stress granules where transcripts are sorted for degradation.…”

Section: Signaling Pathways and Mrna Turnover Controlmentioning

confidence: 99%

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mRNA stability control: a clandestine force in normal and malignant hematopoiesis

Steinman

2007

Leukemia

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This review addresses the scope of influence of mRNA decay on cellular functions and its potential role in normal and malignant hematopoiesis. Evidence is emerging that leukemic oncogenes and hematopoietic cytokines interact with mRNA decay pathways. These pathways can co-regulate functionally related genes through specific motifs in the 3 0 -untranslated region of targeted transcripts. The steps that link external stimuli to transcript turnover are not fully understood, but include subcellular relocalization or post-transcriptional modification of specific transcript-stabilizing or -destabilizing proteins. Improper functioning of these regulators of mRNA turnover can impede normal cellular differentiation or promote cancers. By delineating how subsets of transcripts decay in synchrony during normal hematopoiesis, it may be possible to determine whether this post-transcriptional regulatory pathway is hijacked in leukemogenesis.

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Tristetraprolin (TTP)-14-3-3 Complex Formation Protects TTP from Dephosphorylation by Protein Phosphatase 2a and Stabilizes Tumor Necrosis Factor-α mRNA

Abstract: Tumor necrosis factor (TNF)-

Cited by 176 publications

References 52 publications

MAPKAP Kinase 2 Blocks Tristetraprolin-directed mRNA Decay by Inhibiting CAF1 Deadenylase Recruitment

MAPKAP Kinase 2 Blocks Tristetraprolin-directed mRNA Decay by Inhibiting CAF1 Deadenylase Recruitment

Phosphorylation site analysis of the anti-inflammatory and mRNA-destabilizing protein tristetraprolin

mRNA stability control: a clandestine force in normal and malignant hematopoiesis

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