RESUMO
Posttranscriptional regulatory mechanisms control TNFalpha expression through AU-rich elements in the 3'UTR of its mRNA. This is mediated through Erk and p38 MAP kinase signaling, although the mechanisms involved remain poorly understood. Here, we show that the MAP kinase signal-integrating kinases (Mnks), which are activated by both these pathways, regulate TNFalpha expression in T cells via the 3'UTR. A selective Mnk inhibitor or siRNA-mediated knockdown of Mnk1 inhibits TNFalpha production in T cells, whereas Mnk1 overexpression enhances expression of a reporter construct containing the TNFalpha 3'UTR. We identify ARE binding proteins that are Mnk substrates, such as hnRNP A1, which they phosphorylate at two sites in vitro. hnRNP A1 is phosphorylated in response to T cell activation, and this is blocked by Mnk inhibition. Moreover, Mnk-mediated phosphorylation decreases binding of hnRNP A1 to TNFalpha-ARE in vitro or TNFalpha-mRNA in vivo. Therefore, Mnks are novel players in cytokine regulation and potential new targets for anti-inflammatory therapy.
Assuntos
Ribonucleoproteínas Nucleares Heterogêneas Grupo A-B/metabolismo , Sistema de Sinalização das MAP Quinases/fisiologia , Fator de Necrose Tumoral alfa/biossíntese , Regiões 3' não Traduzidas/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/antagonistas & inibidores , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Genes Reporter , Ribonucleoproteína Nuclear Heterogênea A1 , Ribonucleoproteínas Nucleares Heterogêneas Grupo A-B/antagonistas & inibidores , Humanos , Células Jurkat , Fosforilação , Ligação Proteica/genética , Ligação Proteica/imunologia , Biossíntese de Proteínas/fisiologia , RNA Mensageiro/metabolismo , Fator de Necrose Tumoral alfa/antagonistas & inibidores , Fator de Necrose Tumoral alfa/genética , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
The cap-binding eukaryotic initiation factor eIF4E is phosphorylated by the mitogen-activated protein (MAP) kinase-interacting kinases (Mnk's). Three forms of the Mnk's exist in human cells: Mnk1, Mnk2a, and Mnk2b. These last two are derived from the same gene by alternative splicing and differ only at their C termini. While Mnk2a contains a MAP kinase-binding site in this region, Mnk2b lacks such a sequence and is much less readily activated by MAP kinases in vitro. Expression of Mnk2b in mammalian cells leads to increased phosphorylation of eIF4E, showing that it acts as an eIF4E kinase in vivo. While Mnk2a is cytoplasmic, a substantial amount of Mnk2b is found in the nucleus. Both enzymes contain a stretch of basic residues in their N termini that plays a role in binding to eIF4G and functions as a nuclear localization signal. Binding of eIF4G or nuclear import appears to be regulated by the C terminus of Mnk2a. Furthermore, the MAP kinase-binding site of Mnk2a regulates nuclear entry. Within the nucleus, Mnk2b and certain variants of Mnk2a that are present in the nucleus colocalize with the promyelocytic leukemia protein PML, which also binds to eIF4E.