AU-rich elements (AREs) in the 3' untranslated region (UTR) of unstable mRNAs dictate their degradation. An RNAi-based screen performed in Drosophila S2 cells has revealed that Dicer1, Argonaute1 (Ago1) and Ago2, components involved in microRNA (miRNA) processing and function, are required for the rapid decay of mRNA containing AREs of tumor necrosis factor-alpha. The requirement for Dicer in the instability of ARE-containing mRNA (ARE-RNA) was confirmed in HeLa cells. We further observed that miR16, a human miRNA containing an UAAAUAUU sequence that is complementary to the ARE sequence, is required for ARE-RNA turnover. The role of miR16 in ARE-RNA decay is sequence-specific and requires the ARE binding protein tristetraprolin (TTP). TTP does not directly bind to miR16 but interacts through association with Ago/eiF2C family members to complex with miR16 and assists in the targeting of ARE. miRNA targeting of ARE, therefore, appears to be an essential step in ARE-mediated mRNA degradation.

Phosphorylation of mitogen-activated protein kinases (MAPKs) on specific tyrosine and threonine sites by MAP kinase kinases (MAPKKs) is thought to be the sole activation mechanism. Here, we report an unexpected activation mechanism for p38alpha MAPK that does not involve the prototypic kinase cascade. Rather it depends on interaction of p38alpha with TAB1 [transforming growth factor-beta-activated protein kinase 1 (TAK1)-binding protein 1] leading to autophosphorylation and activation of p38alpha. We detected formation of a TRAF6-TAB1-p38alpha complex and showed stimulus-specific TAB1-dependent and TAB1-independent p38alpha activation. These findings suggest that alternative activation pathways contribute to the biological responses of p38alpha to various stimuli.

We have cloned and characterized a new member of the p38 group of mitogen-activated protein kinases here termed p38␦. Sequence comparisons revealed that p38␦ is approximately 60% identical to the other three p38 isoforms but only 40 -45% to the other mitogen-activated protein kinase family members. It contains the TGY dual phosphorylation site present in all p38 group members and is activated by a group of extracellular stimuli including cytokines and environmental stresses that also activate the other three known p38 isoforms. However, unlike the other p38 isoforms, the kinase activity of p38␦ is not blocked by the pyridinyl imidazole, 4-(4-fluorophenyl)-2-2(4-hydroxyphenyl)-5-(4-pyridyl)-imidazole (identicalto SB202190). p38␦ can be activated by MKK3 and MKK6, known activators of the other isoforms. Nonetheless, in-gel kinase assays provide evidence for additional activators. The data presented herein show that p38␦ has many properties that are similar to those of other p38 group members. Nonetheless important differences exist among the four members of the p38 group of enzymes, and thus each may have highly specific, individual contributions to biologic events involving activation of the p38 pathways.

Members of the MEF2 family of transcription factors bind as homo-and heterodimers to the MEF2 site found in the promoter regions of numerous muscle-specific, growth-or stress-induced genes. We showed previously that the transactivation activity of MEF2C is stimulated by p38 mitogen-activated protein (MAP) kinase. In this study, we examined the potential role of the p38 MAP kinase pathway in regulating the other MEF2 family members. We found that MEF2A, but not MEF2B or MEF2D, is a substrate for p38. Among the four p38 group members, p38 is the most potent kinase for MEF2A. Threonines 312 and 319 within the transcription activation domain of MEF2A are the regulatory sites phosphorylated by p38. Phosphorylation of MEF2A in a MEF2A-MEF2D heterodimer enhances MEF2-dependent gene expression. These results demonstrate that the MAP kinase signaling pathway can discriminate between different MEF2 isoforms and can regulate MEF2-dependent genes through posttranslational activation of preexisting MEF2 protein.The transactivation activity of many transcription factors is regulated by phosphorylation (2). The mitogen-activated protein (MAP) kinase family of serine/threonine kinases has been shown to play important roles in regulating gene expression via transcription factor phosphorylation (5,10,16,38,40,42). Unique structural features, specific activation pathways, and different substrate specificities provide evidence to support the contention that different MAP kinases are independently regulated and control different cellular responses to extracellular stimuli (7,38,40,44).p38 MAP kinase was first identified in studies designed to explore how bacterial endotoxin induces cytokine expression (11,13,23). Following the initial description of p38 (p38␣), three additional isoforms of this MAP kinase group have been cloned and characterized: p38␤ (18), p38␥ (also termed ERK6 or SAPK3) (22,24,30), and p38␦ (also termed SAPK4) (4, 17, 41). p38␣ and p38␤ are sensitive to pyridinyl imidazole derivatives, whereas p38␥ and p38␦ are not (4). In mammalian cells, these closely related p38 isoforms are activated coordinately by a broad panel of stimuli which include physical-chemical stresses and proinflammatory cytokines (17, 36). Two MAP kinase kinases (MKK), MKK3 and MKK6, are the upstream activators of the p38 group MAP kinases (6,12,14,37). Several proteins including transcription factors such as CHOP 10 (GADD153) (42), Sap1 (16), MEF2C (10), enzymes such as cPLA2 (20), and the protein kinases MAPKAPK2/3 (27, 29, 39), MNK1/2 (8, 45), and p38-regulated/activated protein kinase (33) have been shown by us and others to be substrates of p38.We showed that MEF2C, a member of the MEF2 family of transcription factors, is phosphorylated by p38 and that this event regulates the transactivation activity of MEF2C (10). Our studies showed that p38 specifically phosphorylates serine 387 and threonines 293 and 300 within the MEF2C transactivation domain (10). MEF2C phosphorylation by p38 was shown to play an important role in regulation of c-Jun ...

Eukaryotic initiation factor 4E (eIF4E) is a key component of the translational machinery and an important modulator of cell growth and proliferation. The activity of eIF4E is thought to be regulated by interaction with inhibitory binding proteins (4E-BPs) and phosphorylation by mitogen-activated protein (MAP) kinase-interacting kinase (MNK) on Ser209 in response to mitogens and cellular stress. Here we demonstrate that phosphorylation of eIF4E via MNK1 is mediated via the activation of either the Erk or p38 pathway. We further show that expression of active mutants of MNK1 and MNK2 in 293 cells diminishes cap-dependent translation relative to cap-independent translation in a transient reporter assay. The same effect on cap-dependent translation was observed when MNK1 was activated by the Erk or p38 pathway. In line with these findings, addition of recombinant active MNK1 to rabbit reticulocyte lysate resulted in a reduced protein synthesis in vitro, and overexpression of MNK2 caused a decreased rate of protein synthesis in 293 cells. By using CGP 57380, a novel low-molecular-weight kinase inhibitor of MNK1, we demonstrate that eIF4E phosphorylation is not crucial to the formation of the initiation complex, mitogen-stimulated increase in cap-dependent translation, and cell proliferation. Our results imply that activation of MNK by MAP kinase pathways does not constitute a positive regulatory mechanism to cap-dependent translation. Instead, we propose that the kinase activity of MNKs, eventually through phosphorylation of eIF4E, may serve to limit cap-dependent translation under physiological conditions.