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 (GSHbeads or anti-Myc linked to magnetic beads MK21 mediates several p38␣,␤ MAP kinase-dependent processes (for a review, see Ref. 1), demonstrated most clearly by results from targeted disruption of the MK2 gene in mice (2). MK2 (Ϫ,Ϫ) mice have suppressed stress responses. Cellular studies show deficits in motility, chemotaxis, and cytokine production. Macrophages taken from MK2 (Ϫ,Ϫ) mice exhibit normal TNF mRNA induction in response to endotoxin but do not release TNF protein. Cellular TNF protein was markedly decreased in MK2 (Ϫ,Ϫ) macrophages, suggesting a block in production of TNF from TNF mRNA (2). TNF expression is regulated both via mRNA stability and translation (3, 4) but is not completely understood.The p38␣,␤ MAP kinase pathway regulates stability of mRNAs that contain AU-rich elements in their 3Ј-untranslated regions. Examples include TNF, COX-2, interleukin-6, and interleukin-1␤ (4 -6). Evidence chiefly comes from mRNA stabilization caused by transfection of mutationally activated MEK3/MEK6 or by the addition of agents that activate p38 MAPK and conversely from destabilization caused by the addition of a p38␣,␤ MAP kinase inhibitor. Since p38␣,␤ is required to activate MK2, these experiments do not dissect contributions from MK2. Studies in MK2 (Ϫ,Ϫ) cells suggest that MK2 regulates stability of some cytokine mRNAs (2, 4). Lasa et al. (5) first reported that expression of a mutant of MK2 with constitutive activity stabilized COX-2 mRNA in the presence of SB2035780 and that expression of a kinase-defective MK2 blocked the stabilization induced by activated MEK6, arguing that MK2 is necessary and sufficient to induce stabilization of at least the COX-2 mRNA (5). How MK2 regulates cytokine production post-transcriptionally is unknown. Mahtani et al. (7) reported that tristetraprolin (TTP) is an in vitro substrate for MK2, motivating the detailed studies we describe.TTP (for a review, see Ref. 8) destabilizes class II AU-rich elements and is the prototype for a non-zinc finger class of nucleic acid-binding proteins. Destabilization requires integrity of the TTP tandem Cys 3 His RNA binding domains that coordinate zinc in a disklike structure (9 -11). TTP-null mice exhibit many defects including inflammatory arthritis and systemic lupus erythematosis-like symptoms attributed to increased production of TNF (12).TTP is phosphorylated in cells treated with growth factors or cytokines. Phosphorylation occurs at more than one site evident by the appearance of two distinct slower migrating forms of TTP on gels after stimulation that are reversed by phosphatase treatment (7, 13). TTP under...

Regulation of messenger RNA stability by AU-rich elements is an important means of regulating genes induced by growth factors and cytokines. Nup475 (also known as tristetraprolin, or TIS11) is the prototype for a family of zinc-binding Cys 3 His motif proteins required for proper regulation of tumor necrosis factor mRNA stability in macrophages. We developed an Escherichia coli expression system to produce soluble Nup475 protein in quantity to study its RNA binding properties. Nup475 protein bound a tumor necrosis factor AU-rich element over a broad range of pH and salt concentrations by RNA gel shift. This binding was inhibited by excess zinc metal, providing a potential mechanism for previous reports of zinc stabilization of AU-rich element (ARE) containing messenger RNAs. Immobilized Nup475 protein was used to select its optimal binding site by RNA SELEX and revealed a strong preference for the extended sequence UUAUUUAUU, rather than a simple AUUUA motif. These findings were confirmed by sitedirected mutagenesis of the tumor necrosis factor ARE and RNA gel shifts on c-fos, interferon-␥, and interferon-␤ ARE fragments. A weaker binding activity toward adenine-rich sites, such as a poly(A) tail RNA fragment, can partially disrupt the Nup475-tumor necrosis factor AU-rich element complex.

The MNK kinases are downstream of both the p38 and ERK MAP kinase pathways and act to increase gene expression. MNK inhibition using the compound CGP57380 has recently been reported to inhibit tumor necrosis factor (TNF) production in macrophage cell lines stimulated with Escherichia coli lipopolysaccharide (LPS). However, the range of receptors that signal through the MNK kinases and the extent of the resultant cytokine response are not known. We found that TNF production was inhibited in RAW264.7 macrophage cells by CGP57380 in a dose-responsive manner with agonists for Toll-like receptor (TLR) 2 (HKLM), TLR4 (Salmonella LPS), TLR6/2 (FSL), TLR7 (imiquimod), and TLR9 (CpG DNA). CGP57380 also inhibited the peak of TNF mRNA production and increased the rate of TNF mRNA decay, effects not due to the destabilizing RNA binding protein tristetraprolin (TTP). Similar to its effects on TNF, CGP57380 caused dose-responsive inhibition of TTP production from stimulation with either LPS or CpG DNA. MNK inhibition also blocked IL-6 but permitted IL-10 production in response to LPS. Studies using bone marrow-derived macrophages (BMDM) isolated from a spontaneous mouse model of Crohn's disease-like ileitis (SAMP1/YitFc strain) revealed significant inhibition by CGP57380 of the proinflammatory cytokines TNF, IL-6, and monocyte chemoattractant protein-1 at 4 and 24 h after LPS stimulation. IL-10 production was higher in CGP53870-treated BMDM at 4 h but was similar to the controls by 24 h. Taken together, these data demonstrate that MNK kinases signal through a variety of TLR agonists and mediate a potent innate, proinflammatory cytokine response.

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Tristetraprolin (TTP) is the prototype for a family of RNA binding proteins that bind the tumor necrosis factor (TNF) messenger RNA AU-rich element (ARE), causing deadenylation of the TNF poly(A) tail, RNA decay, and silencing of TNF protein production. Using mass spectrometry sequencing we identified poly(A) binding proteins-1 and -4 (PABP1 and PABP4) in high abundance and good protein coverage from TTP immunoprecipitates. PABP1 significantly enhanced TNF ARE binding by RNA EMSA and prevented TTP-initiated deadenylation in an in vitro macrophage assay of TNF poly(A) stability. Neomycin inhibited TTP-promoted deadenylation at concentrations shown to inhibit the deadenylases poly(A) ribonuclease and CCR4. Stably transfected RAW264.7 macrophages overexpressing PABP1 do not oversecrete TNF; instead they upregulate TTP protein without increasing TNF protein production. The PABP1 inhibition of deadenylation initiated by TTP does not require the poly(A) binding regions in RRM1 and RRM2, suggesting a more complicated interaction than simple masking of the poly(A) tail from a 3'-exonuclease. Like TTP, PABP1 is a substrate for p38 MAP kinase. Finally, PABP1 stabilizes cotransfected TTP in 293T cells and prevents the decrease in TTP levels seen with p38 MAP kinase inhibition. These findings suggest several levels of functional antagonism between TTP and PABP1 that have implications for regulation of unstable mRNAs like TNF.