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

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)4·CCR4-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 RNA-independent 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.     

MAPKAP kinase 2 phosphorylates tristetraprolin on in vivo sites including Ser178, a site required for 14-3-3 binding

MAPKAP kinase 2 (MK2) is required for tumor necrosis factor synthesis. Tristetraprolin (TTP) binds to the 3'-untranslated region of tumor necrosis factor mRNA and regulates its fate. We identified in vitro and in vivo phosphorylation sites in TTP using nanoflow high pressure liquid chromatography microelectrospray ionization tandem mass spectrometry and novel methods for direct digestion of TTP bound to affinity matrices (GSH-beads or anti-Myc linked to magnetic beads). MK2Delta3B, activated in Escherichia coli by p38alpha, phosphorylates TTP in vitro at major sites Ser(52) and Ser(178) (>10-fold in abundance) as well as at several minor sites that were detected after enriching for phosphopeptides with immobilized metal affinity chromatography. MK2 phosphorylation of TTP creates a functional 14-3-3 binding site. In cells, TTP was phosphorylated at Ser(52), Ser(178), Thr(250), and Ser(316) and at SP sites in a cluster (Ser(80)/Ser(82)/Ser(85)). Anisomycin treatment of NIH 3T3 cells increased phosphorylation of Ser(52) and Ser(178). Overexpression of MK2 sufficed to increase phosphorylation of Ser(52) and Ser(178) but not Ser(80)/Ser(82)/Ser(85) or Thr(250). Thus, Ser(52) and Ser(178) are putative MK2 sites in vivo. Identified phosphosite(s) may be biologic switches controlling mRNA stability and translation.     

A role for transportin in deposition of TTP to cytoplasmic RNA granules and mRNA decay

Importin-β family members, which shuttle between the nucleus and the cytoplasm, are essential for nucleocytoplasmic transport of macromolecules. We attempted to explore whether importin-β family proteins change their cellular localization in response to environmental change. In this report, we show that transportin (TRN) was minimally detected in cytoplasmic processing bodies (P-bodies) under normal cell conditions but largely translocated to stress granules (SGs) in stressed cells. Fluorescence recovery after photobleaching analysis indicated that TRN moves rapidly in and out of cytoplasmic granules. Depletion of TRN greatly enhanced P-body formation but did not affect the number or size of SGs, suggesting that TRN or its cargo(es) participates in cellular function of P-bodies. Accordingly, TRN associated with tristetraprolin (TTP) and its AU-rich element (ARE)-containing mRNA substrates. Depletion of TRN increased the number of P-bodies and stabilized ARE-containing mRNAs, as observed with knockdown of the 5′–3′ exonuclease Xrn1. Moreover, depletion of TRN retained TTP in P-bodies and meanwhile reduced the fraction of mobile TTP to SGs. Therefore, our data together suggest that TRN plays a role in trafficking of TTP between the cytoplasmic granules and whereby modulates the stability of ARE-containing mRNAs.     

G3BP–Caprin1–USP10 complexes mediate stress granule condensation and associate with 40S subunits

Mammalian stress granules (SGs) contain stalled translation preinitiation complexes that are assembled into discrete granules by specific RNA-binding proteins such as G3BP. We now show that cells lacking both G3BP1 and G3BP2 cannot form SGs in response to eukaryotic initiation factor 2α phosphorylation or eIF4A inhibition, but are still SG-competent when challenged with severe heat or osmotic stress. Rescue experiments using G3BP1 mutants show that phosphomimetic G3BP1-S149E fails to rescue SG formation, whereas G3BP1-F33W, a mutant unable to bind G3BP partner proteins Caprin1 or USP10, rescues SG formation. Caprin1/USP10 binding to G3BP is mutually exclusive: Caprin binding promotes, but USP10 binding inhibits, SG formation. G3BP interacts with 40S ribosomal subunits through its RGG motif, which is also required for G3BP-mediated SG formation. We propose that G3BP mediates the condensation of SGs by shifting between two different states that are controlled by the phosphorylation of S149 and by binding to Caprin1 or USP10.     

Evidence That Ternary Complex (eIF2-GTP-tRNAiMet)–Deficient Preinitiation Complexes Are Core Constituents of Mammalian Stress Granules

Environmental stress-induced phosphorylation of eIF2alpha inhibits protein translation by reducing the availability of eIF2-GTP-tRNA(i)Met, the ternary complex that joins initiator tRNA(Met) to the 43S preinitiation complex. The resulting untranslated mRNA is dynamically routed to discrete cytoplasmic foci known as stress granules (SGs), a process requiring the related RNA-binding proteins TIA-1 and TIAR. SGs appear to be in equilibrium with polysomes, but the nature of this relationship is obscure. We now show that most components of the 48S preinitiation complex (i.e., small, but not large, ribosomal subunits, eIF3, eIF4E, eIF4G) are coordinately recruited to SGs in arsenite-stressed cells. In contrast, eIF2 is not a component of newly assembled SGs. Cells expressing a phosphomimetic mutant (S51D) of eIF2alpha assemble SGs of similar composition, confirming that the recruitment of these factors is a direct consequence of blocked translational initiation and not due to other effects of arsenite. Surprisingly, phospho-eIF2alpha is recruited to SGs that are disassembling in cells recovering from arsenite-induced stress. We discuss these results in the context of a translational checkpoint model wherein TIA and eIF2 are functional antagonists of translational initiation, and in which lack of ternary complex drives SG assembly.     

14-3-3epsilon inhibits MK5-mediated cell migration by disrupting F-actin polymerization

The signal pathway by which 14-3-3epsilon inhibits cell migration induced by MAPK-activated protein kinase 5 (MK5) was investigated in cultured HeLa cells. Both in vivo and in vitro analyses have revealed that 14-3-3epsilon interacts with MK5. 14-3-3epsilon bound to MK5 inhibits the phosphorylation of HSP27, a known substrate of MK5. Disturbance of actin cytoskeleton organization by 14-3-3epsilon was shown in transfected cells transiently expressing 14-3-3epsilon as well as established cells stably expressing 14-3-3epsilon. Moreover, overexpression of 14-3-3epsilon resulted in the inhibition of cell migration induced by MK5 overexpression or TNFalpha treatment. Our results suggest that 14-3-3epsilon bound to MK5 inhibits cell migration by inhibiting the phosphorylation of HSP27 whose phosphorylation regulates F-actin polymerization, actin cytoskeleton organization and subsequent actinfilament dynamics.     

Stress granule assembly is mediated by prion-like aggregation of TIA-1

TIA-1 is an RNA binding protein that promotes the assembly of stress granules (SGs), discrete cytoplasmic inclusions into which stalled translation initiation complexes are dynamically recruited in cells subjected to environmental stress. The RNA recognition motifs of TIA-1 are linked to a glutamine-rich prion-related domain (PRD). Truncation mutants lacking the PRD domain do not induce spontaneous SGs and are not recruited to arsenite-induced SGs, whereas the PRD forms aggregates that are recruited to SGs in low-level-expressing cells but prevent SG assembly in high-level-expressing cells. The PRD of TIA-1 exhibits many characteristics of prions: concentration-dependent aggregation that is inhibited by the molecular chaperone heat shock protein (HSP)70; resistance to protease digestion; sequestration of HSP27, HSP40, and HSP70; and induction of HSP70, a feedback regulator of PRD disaggregation. Substitution of the PRD with the aggregation domain of a yeast prion, SUP35-NM, reconstitutes SG assembly, confirming that a prion domain can mediate the assembly of SGs. Mouse embryomic fibroblasts (MEFs) lacking TIA-1 exhibit impaired ability to form SGs, although they exhibit normal phosphorylation of eukaryotic initiation factor (eIF)2alpha in response to arsenite. Our results reveal that prion-like aggregation of TIA-1 regulates SG formation downstream of eIF2alpha phosphorylation in response to stress.     

Cytoplasmic localization of tristetraprolin involves 14-3-3-dependent and -independent mechanisms

The immediate early gene tristetraprolin (TTP) is induced transiently in many cell types by numerous extracellular stimuli. TTP encodes a zinc finger protein that can bind and destabilize mRNAs that encode tumor necrosis factor-alpha (TNFalpha) and other cytokines. We hypothesize that TTP also has a broader role in growth factor-responsive pathways. In support of this model, we have previously determined that TTP induces apoptosis through the mitochondrial pathway, analogously to certain oncogenes and other immediate-early genes, and that TTP sensitizes cells to the pro-apoptotic signals of TNFalpha. In this study, we show that TTP and the related proteins TIS11b and TIS11d bind specifically to 14-3-3 proteins and that individual 14-3-3 isoforms preferentially bind to different phosphorylated TTP species. 14-3-3 binding does not appear to inhibit or promote induction of apoptosis by TTP but is one of multiple mechanisms that localize TTP to the cytoplasm. Our results provide the first example of 14-3-3 interacting functionally with an RNA binding protein and binding in vivo to a Type II 14-3-3 binding site. They also suggest that 14-3-3 binding is part of a complex network of stimuli and interactions that regulate TTP function.     

Stress granules contribute to α-globin homeostasis in differentiating erythroid cells

Hemoglobin is the major biosynthetic product of developing erythroid cells. Assembly of hemoglobin requires the balanced production of globin proteins and the oxygen-carrying heme moiety. The heme-regulated inhibitor kinase (HRI) participates in this process by phosphorylating eIF2α and inhibiting the translation of globin proteins when levels of free heme are limiting. HRI is also activated in erythroid cells subjected to oxidative stress. Phospho-eIF2α-mediated translational repression induces the assembly of stress granules (SG), cytoplasmic foci that harbor untranslated mRNAs and promote the survival of cells subjected to adverse environmental conditions. We have found that differentiating erythroid, but not myelomonocytic or megakaryocytic, murine and human progenitor cells assemble SGs, in vitro and in vivo. Targeted knockdown of HRI or G3BP, a protein required for SG assembly, inhibits spontaneous and arsenite-induced assembly of SGs in erythroid progenitor cells. This is accompanied by reduced α-globin production and increased apoptosis suggesting that G3BP+ SGs facilitate the survival of developing erythroid cells.     

Structure/function analysis of tristetraprolin (TTP): p38 stress-activated protein kinase and lipopolysaccharide stimulation do not alter TTP function

Tristetraprolin (TTP) is the only trans-acting factor shown to be capable of regulating AU-rich element-dependent mRNA turnover at the level of the intact animal; however, the mechanism by which TTP mediated RNA instability is unknown. Using an established model system, we performed structure/function analysis with TTP as well as examined the current hypothesis that TTP function is regulated by p38-MAPKAP kinase 2 (MK2) activation. Deletion of either the N- or C-terminal domains inhibited TTP function. Extensive mutagenesis, up to 16%, of serines and threonines, some of which were predicted to mediate proteasomal targeting, did not alter human TTP function. Mutation of the conserved MK2 phosphorylation sites enhanced human TTP function in both resting and p38-stress-activated protein kinase-MK2-activated cells. However, p38-stress-activated protein kinase-MK2 activation did not alter the activity of either wild-type or mutant TTP. TTP localized to the stress granules, with arsenite treatment reducing this localization. In contrast, arsenite treatment enhanced stress granule localization of the MK2 mutant, consistent with the involvement of additional pathways regulating this event. Finally, we determined that, in response to LPS stimulation, human TTP moves onto the polysomes, and this movement occurs in the absence of 14-3-3. Taken together, these data indicate that, although p38 activation alters TTP entry into the stress granule, it does not alter TTP function. Moreover, the interaction of TTP with 14-3-3, which may limit entry into the stress granule, is not involved in the downstream message stabilization events.     

Sequestration of G3BP coupled with efficient translation inhibits stress granules in Semliki Forest virus infection

Dynamic, mRNA-containing stress granules (SGs) form in the cytoplasm of cells under environmental stresses, including viral infection. Many viruses appear to employ mechanisms to disrupt the formation of SGs on their mRNAs, suggesting that they represent a cellular defense against infection. Here, we report that early in Semliki Forest virus infection, the C-terminal domain of the viral nonstructural protein 3 (nsP3) forms a complex with Ras-GAP SH3-domain–binding protein (G3BP) and sequesters it into viral RNA replication complexes in a manner that inhibits the formation of SGs on viral mRNAs. A viral mutant carrying a C-terminal truncation of nsP3 induces more persistent SGs and is attenuated for propagation in cell culture. Of importance, we also show that the efficient translation of viral mRNAs containing a translation enhancer sequence also contributes to the disassembly of SGs in infected cells. Furthermore, we show that the nsP3/G3BP interaction also blocks SGs induced by other stresses than virus infection. This is one of few described viral mechanisms for SG disruption and underlines the role of SGs in antiviral defense.     

Hydrogen peroxide induces stress granule formation independent of eIF2α phosphorylation

In cells exposed to environmental stress, inhibition of translation initiation conserves energy for the repair of cellular damage. Untranslated mRNAs that accumulate in these cells move to discrete cytoplasmic foci known as stress granules (SGs). The assembly of SGs helps cells to survive under adverse environmental conditions. We have analyzed the mechanism by which hydrogen peroxide (H(2)O(2))-induced oxidative stress inhibits translation initiation and induces SG assembly in mammalian cells. Our data indicate that H(2)O(2) inhibits translation and induces the assembly of SGs. The assembly of H(2)O(2)-induced SGs is independent of the phosphorylation of eIF2α, a major trigger of SG assembly, but requires remodeling of the cap-binding eIF4F complex. Moreover, H(2)O(2)-induced SGs are compositionally distinct from canonical SGs, and targeted knockdown of eIF4E, a protein required for canonical translation initiation, inhibits H(2)O(2)-induced SG assembly. Our data reveal new aspects of translational regulation induced by oxidative insults.     

Polyamines inhibit the assembly of stress granules in normal intestinal epithelial cells regulating apoptosis

Polyamines regulate multiple signaling pathways and are implicated in many aspects of cellular functions, but the exact molecular processes governed by polyamines remain largely unknown. In response to environmental stress, repression of translation is associated with the assembly of stress granules (SGs) that contain a fraction of arrested mRNAs and are thought to function as mRNA storage. Here we show that polyamines modulate the assembly of SGs in normal intestinal epithelial cells (IECs) and that induced SGs following polyamine depletion are implicated in the protection of IECs against apoptosis. Increasing the levels of cellular polyamines by ectopic overexpression of the ornithine decarboxylase gene decreased cytoplasmic levels of SG-signature constituent proteins eukaryotic initiation factor 3b and T-cell intracellular antigen-1 (TIA-1)-related protein and repressed the assembly of SGs induced by exposure to arsenite-induced oxidative stress. In contrast, depletion of cellular polyamines by inhibiting ornithine decarboxylase with α-difluoromethylornithine increased cytoplasmic eukaryotic initiation factor 3b and TIA-1 related protein abundance and enhanced arsenite-induced SG assembly. Polyamine-deficient cells also exhibited an increase in resistance to tumor necrosis factor-α/cycloheximide-induced apoptosis, which was prevented by inhibiting SG formation with silencing SG resident proteins Sort1 and TIA-1. These results indicate that the elevation of cellular polyamines represses the assembly of SGs in normal IECs and that increased SGs in polyamine-deficient cells are crucial for increased resistance to apoptosis.     

eIF5A Promotes Translation Elongation,Polysome Disassembly and Stress Granule Assembly

Stress granules (SGs) are cytoplasmic foci at which untranslated mRNAs accumulate in cells exposed to environmental stress. We have identified ornithine decarboxylase (ODC), an enzyme required for polyamine synthesis, and eIF5A, a polyamine (hypusine)-modified translation factor, as proteins required for arsenite-induced SG assembly. Knockdown of deoxyhypusine synthase (DHS) or treatment with a deoxyhypusine synthase inhibitor (GC7) prevents hypusine modification of eIF5A as well as arsenite-induced polysome disassembly and stress granule assembly. Time-course analysis reveals that this is due to a slowing of stress-induced ribosome run-off in cells lacking hypusine-eIF5A. Whereas eIF5A only marginally affects protein synthesis under normal conditions, it is required for the rapid onset of stress-induced translational repression. Our results reveal that hypusine-eIF5A-facilitated translation elongation promotes arsenite-induced polysome disassembly and stress granule assembly in cells subjected to adverse environmental conditions.     

Inhibition of the ubiquitin-proteasome system induces stress granule formation

The inhibition of the ubiquitin-dependent proteasome system (UPS) via specific drugs is one type of approach used to combat cancer. Although it has been suggested that UPS inhibition prevents the rapid decay of AU-rich element (ARE)-containing messages, very little is known about the cellular mechanisms leading to this effect. Here we establish a link between the inhibition of UPS activity, the formation of cytoplasmic stress granules (SGs), and mRNA metabolism. The assembly of the SGs requires the phosphorylation of the translation initiation factor eIF2alpha by a mechanism involving the stress kinase GCN2. On prolonged UPS inhibition and despite the maintenance of eIF2alpha phosphorylation, SGs disassemble and translation recovers in an Hsp72 protein-dependent manner. The formation of these SGs coincides with the disassembly of processing bodies (PBs), known as mRNA decay entities. As soon as the SGs assemble, they recruit ARE-containing messages such as p21(cip1) mRNA, which are stabilized under these conditions. Hence, our findings suggest that SGs could be considered as one of the players that mediate the early response of the cell to proteasome inhibitors by interfering temporarily with mRNA decay pathways.     

The p38/MK2-Driven Exchange between Tristetraprolin and HuR Regulates AU–Rich Element–Dependent Translation

TNF expression of macrophages is under stringent translational control that depends on the p38 MAPK/MK2 pathway and the AU–rich element (ARE) in the TNF mRNA. Here, we elucidate the molecular mechanism of phosphorylation-regulated translation of TNF. We demonstrate that translation of the TNF-precursor at the ER requires expression of the ARE–binding and -stabilizing factor human antigen R (HuR) together with either activity of the p38 MAPK/MK2 pathway or the absence of the ARE-binding and -destabilizing factor tristetraprolin (TTP). We show that phosphorylation of TTP by MK2 decreases its affinity to the ARE, inhibits its ability to replace HuR, and permits HuR-mediated initiation of translation of TNF mRNA. Since translation of TTP''s own mRNA is also regulated by this mechanism, an intrinsic feedback control of the inflammatory response is ensured. The phosphorylation-regulated TTP/HuR exchange at target mRNAs provides a reversible switch between unstable/non-translatable and stable/efficiently translated mRNAs.