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.

Mitogen-activated protein kinase (MAPK)-activated protein kinase 2 (MK2) is activated upon stress by p38 MAPK␣ and -␤, which bind to a basic docking motif in the C terminus of MK2 and which subsequently phosphorylate its regulatory sites. As a result of activation MK2 is exported from the nucleus to the cytoplasm and cotransports active p38 MAPK to this compartment. Here we show that the amount of p38 MAPK is significantly reduced in cells and tissues lacking MK2, indicating a stabilizing effect of MK2 for p38. Using a murine knockout model, we have previously shown that elimination of MK2 leads to a dramatic reduction of tumor necrosis factor (TNF) production in response to lipopolysaccharide. To further elucidate the role of MK2 in p38 MAPK stabilization and in TNF biosynthesis, we analyzed the ability of two MK2 isoforms and several MK2 mutants to restore both p38 MAPK protein levels and TNF biosynthesis in macrophages. We show that MK2 stabilizes p38 MAPK through its C terminus and that MK2 catalytic activity does not contribute to this stabilization. Importantly, we demonstrate that stabilizing p38 MAPK does not restore TNF biosynthesis. TNF biosynthesis is only restored with MK2 catalytic activity. We further show that, in MK2-deficient macrophages, formation of filopodia in response to extracellular stimuli is reduced. In addition, migration of MK2-deficient mouse embryonic fibroblasts (MEFs) and smooth muscle cells on fibronectin is dramatically reduced. Interestingly, reintroducing catalytic MK2 activity into MEFs alone is not sufficient to revert the migratory phenotype of these cells. In addition to catalytic activity, the proline-rich N-terminal region is necessary for rescuing the migratory phenotype. These data indicate that catalytic activity of MK2 is required for both cytokine production and cell migration. However, the proline-rich MK2 N terminus provides a distinct role restricted to cell migration.The stress-activated kinase mitogen-activated protein kinase (MAPK)-activated protein kinase 2 (MK2) is a direct substrate of p38 MAPK␣ and -␤ (also designated stress-activated protein kinase 2a and 2b). Phosphorylation of MK2 by p38 MAPK serves a dual function. First, it results in the activation of MK2 kinase activity, which in turn leads to the phosphorylation of substrates of MK2 such as small heat shock protein Hsp25/27, tyrosine hydroxylase, and leukocyte-specific protein 1. In addition, MK2 determines the subcellular localization of p38 MAPK. Phosphorylation of MK2 by p38 MAPK unmasks a nuclear export signal (NES) in the C-terminal part of the molecule (4). This unmasking is a prerequisite for nucleocytoplasmic transport of MK2, which in turn coexports activated p38 MAPK from the nucleus to the cytoplasm. Notably, MK2 catalytic activity is not required for cotransport of p38 (1). Using a targeted deletion of MK2, we have shown that MK2 is posttranscriptionally required for the lipopolysaccharide (LPS)-induced production of several cytokines including tumor necrosis factor (TNF) and interleuki...

Extracellular-regulated kinase 3 (ERK3, MAPK6) is an atypical member of the ERKs, lacking the threonine and tyrosine residues in the activation loop, carrying a unique C-terminal extension and being mainly regulated by its own protein stability and/or by autophosphorylation. Here we show that ERK3 specifically interacts with the MAPKactivated protein kinase 5 (MK5 or PRAK) in vitro and in vivo. Expression of ERK3 in mammalian cells leads to nuclear-cytoplasmic translocation and activation of MK5 and to phosphorylation of both ERK3 and MK5. Remarkably, activation of MK5 is independent of ERK3 enzymatic activity, but depends on its own catalytic activity as well as on a region in the C-terminal extension of ERK3. In mouse embryonic development, mRNA expression patterns of ERK3 and MK5 suggest spatiotemporal coexpression of both kinases. Deletion of MK5 leads to strong reduction of ERK3 protein levels and embryonic lethality at about stage E11, where ERK3 expression in wild-type mice is maximum, indicating a role of this signalling module in development.

RNA-binding proteins (RBPs) facilitate post-transcriptional control of eukaryotic gene expression at multiple levels. The RBP tristetraprolin (TTP/Zfp36) is a signal-induced phosphorylated anti-inflammatory protein guiding unstable mRNAs of pro-inflammatory proteins for degradation and preventing translation. Using iCLIP, we have identified numerous mRNA targets bound by wild-type TTP and by a non-MK2-phosphorylatable TTP mutant (TTP-AA) in 1 h LPS-stimulated macrophages and correlated their interaction with TTP to changes at the level of mRNA abundance and translation in a transcriptome-wide manner. The close similarity of the transcriptomes of TTP-deficient and TTP-expressing macrophages upon short LPS stimulation suggested an effective inactivation of TTP by MK2, whereas retained RNA-binding capacity of TTP-AA to 3′UTRs caused profound changes in the transcriptome and translatome, altered NF-κB-activation and induced cell death. Increased TTP binding to the 3′UTR of feedback inhibitor mRNAs, such as Ier3, Dusp1 or Tnfaip3, in the absence of MK2-dependent TTP neutralization resulted in a strong reduction of their protein synthesis contributing to the deregulation of the NF-κB-signaling pathway. Taken together, our study uncovers a role of TTP as a suppressor of feedback inhibitors of inflammation and highlights the importance of fine-tuned TTP activity-regulation by MK2 in order to control the pro-inflammatory response.

MK5 (mitogen-activated protein kinase [MAPK]-activated protein kinase 5), also designated PRAK (p38-regulated and -activated kinase), was deleted from mice by homologous recombination. Although no MK5 full-length protein and kinase activity was detected in the MK5 knockout mice, the animals were viable and fertile and did not display abnormalities in tissue morphology or behavior. In addition, these mice did not show increased resistance to endotoxic shock or decreased lipopolysaccharide-induced cytokine production. Hence, MK5 deletion resulted in a phenotype very different from the complex inflammation-impaired phenotype of mice deficient in MK2, although MK2 and MK5 exhibit evolutional, structural, and apparent extensive functional similarities. To explain this discrepancy, we used wild-type cells and embryonic fibroblasts from both MK2 and MK5 knockout mice as controls to reexamine the mechanism of activation, the interaction with endogenous p38 MAPK, and the substrate specificity of both enzymes. In contrast to MK2, which shows interaction with and chaperoning properties for p38 MAPK and which is activated by extracellular stresses such as arsenite or sorbitol treatment, endogenous MK5 did not show these properties. Furthermore, endogenous MK5 is not able to phosphorylate Hsp27 in vitro and in vivo. We conclude that the differences between the phenotypes of MK5-and MK2-deficient mice result from clearly different functional properties of both enzymes.In mouse and human genomes, a group of three mitogenactivated protein kinase (MAPK)-activated protein (MAP-KAP) kinases (MKs), MK2, MK3/3pK, and MK5/PRAK (p38-regulated and -activated kinase), has been identified and the three kinases have been assigned neighboring positions in the phylogenetic tree (14). These three kinases also show apparent functional similarities: they carry a nuclear localization signal, show nuclear localization in resting cells (3,17), and are exported from the nucleus upon activation (1,3,23,26). Furthermore, the three kinases seem to have similar positions in the stress-activated p38 MAPK signaling pathway, i.e., all three seem to bind to p38 MAPK-␣/SAPK2a (15, 26) and are phosphorylated and activated by p38 MAPK-␣ and -␤/ 15,19,21). The substrate specificities of these kinases also seem very similar. The small heat shock protein Hsp25/27 has been described as an efficient substrate for all three kinases in vitro (15,19,25) and also for MK2 in vivo (10).Taking into account these similarities and the ubiquitous expression of these enzymes in the different cells and tissues analyzed (4,19,24), it was rather surprising that mice lacking only one of the three kinases, MK2, showed a clear phenotype: deletion of the gene for MK2 led to a defect in lipopolysaccharide (LPS)-induced biosynthesis of cytokines such as tumor necrosis factor (TNF), interleukin-6 (IL-6), and gamma interferon (IFN-␥) (10) and also to changes in cell migration (7,11). Although the lack in catalytic activity of MK2 was responsible for the defects in cytokine biosynthesi...