RESUMO
Phosphorylation of eukaryotic translation initiation factor 2 alpha (eIF2α) by the kinase GCN2 attenuates protein synthesis during amino acid starvation in yeast, whereas in mammals a family of related eIF2α kinases regulate translation in response to a variety of stresses. Unlike single-celled eukaryotes, mammals also possess two specific eIF2α phosphatases, PPP1R15a and PPP1R15b, whose combined deletion leads to a poorly understood early embryonic lethality. We report the characterisation of the first non-mammalian eIF2α phosphatase and the use of Drosophila to dissect its role during development. The Drosophila protein demonstrates features of both mammalian proteins, including limited sequence homology and association with the endoplasmic reticulum. Of note, although this protein is not transcriptionally regulated, its expression is controlled by the presence of upstream open reading frames in its 5'UTR, enabling induction in response to eIF2α phosphorylation. Moreover, we show that its expression is necessary for embryonic and larval development and that this is to oppose the inhibitory effects of GCN2 on anabolic growth.
Assuntos
Proteínas de Drosophila/metabolismo , Drosophila melanogaster/crescimento & desenvolvimento , Fator de Iniciação 2 em Eucariotos/metabolismo , Proteínas Quinases/metabolismo , Proteína Fosfatase 1/metabolismo , Regiões 5' não Traduzidas/genética , Sequência de Aminoácidos , Animais , Animais Geneticamente Modificados , Células COS , Chlorocebus aethiops , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Embrião não Mamífero , Retículo Endoplasmático/metabolismo , Estresse do Retículo Endoplasmático , Fator de Iniciação 2 em Eucariotos/genética , Células HEK293 , Humanos , Dados de Sequência Molecular , Fosforilação/genética , Proteínas Quinases/genética , Proteína Fosfatase 1/genética , Processamento Pós-Transcricional do RNA/genética , Homologia de Sequência de AminoácidosRESUMO
Inhibition of oncogenic transcriptional programs is a promising therapeutic strategy. A substituted tricyclic benzimidazole, SEL120-34A, is a novel inhibitor of Cyclin-dependent kinase 8 (CDK8), which regulates transcription by associating with the Mediator complex. X-ray crystallography has shown SEL120-34A to be a type I inhibitor forming halogen bonds with the protein's hinge region and hydrophobic complementarities within its front pocket. SEL120-34A inhibits phosphorylation of STAT1 S727 and STAT5 S726 in cancer cells in vitro. Consistently, regulation of STATs- and NUP98-HOXA9- dependent transcription has been observed as a dominant mechanism of action in vivo. Treatment with the compound resulted in a differential efficacy on AML cells with elevated STAT5 S726 levels and stem cell characteristics. In contrast, resistant cells were negative for activated STAT5 and revealed lineage commitment. In vivo efficacy in xenotransplanted AML models correlated with significant repression of STAT5 S726. Favorable pharmacokinetics, confirmed safety and in vivo efficacy provide a rationale for the further clinical development of SEL120-34A as a personalized therapeutic approach in AML.