RESUMO
DENR and MCTS1 have been identified as oncogenes in several different tumor entities. The heterodimeric DENR·MCTS1 protein complex promotes translation of mRNAs containing upstream Open Reading Frames (uORFs). We show here that DENR is phosphorylated on Serine 73 by Cyclin B/CDK1 and Cyclin A/CDK2 at the onset of mitosis, and then dephosphorylated as cells exit mitosis. Phosphorylation of Ser73 promotes mitotic stability of DENR protein and prevents its cleavage at Asp26. This leads to enhanced translation of mRNAs involved in mitosis. Indeed, we find that roughly 40% of all mRNAs with elevated translation in mitosis are DENR targets. In the absence of DENR or of Ser73 phosphorylation, cells display elevated levels of aberrant mitoses and cell death. This provides a mechanism how the cell cycle regulates translation of a subset of mitotically relevant mRNAs during mitosis.
Assuntos
Proteína Quinase CDC2/metabolismo , Ciclina A/metabolismo , Ciclina B/metabolismo , Quinase 2 Dependente de Ciclina/metabolismo , Fatores de Iniciação em Eucariotos/metabolismo , Western Blotting , Proteína Quinase CDC2/genética , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Divisão Celular/genética , Linhagem Celular Tumoral , Ciclina A/genética , Ciclina B/genética , Quinase 2 Dependente de Ciclina/genética , Fatores de Iniciação em Eucariotos/genética , Células HeLa , Humanos , Células MCF-7 , Mitose/genética , Proteínas Oncogênicas/genética , Proteínas Oncogênicas/metabolismo , Fases de Leitura Aberta/genética , Fosforilação , Biossíntese de Proteínas/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Serina/genética , Serina/metabolismoRESUMO
Translation efficiency varies considerably between different mRNAs, thereby impacting protein expression. Translation of the stress response master-regulator ATF4 increases upon stress, but the molecular mechanisms are not well understood. We discover here that translation factors DENR, MCTS1 and eIF2D are required to induce ATF4 translation upon stress by promoting translation reinitiation in the ATF4 5'UTR. We find DENR and MCTS1 are only needed for reinitiation after upstream Open Reading Frames (uORFs) containing certain penultimate codons, perhaps because DENRâ¢MCTS1 are needed to evict only certain tRNAs from post-termination 40S ribosomes. This provides a model for how DENR and MCTS1 promote translation reinitiation. Cancer cells, which are exposed to many stresses, require ATF4 for survival and proliferation. We find a strong correlation between DENRâ¢MCTS1 expression and ATF4 activity across cancers. Furthermore, additional oncogenes including a-Raf, c-Raf and Cdk4 have long uORFs and are translated in a DENRâ¢MCTS1 dependent manner.
Assuntos
Fator 4 Ativador da Transcrição/genética , Fatores de Iniciação em Eucariotos/metabolismo , Biossíntese de Proteínas , Ribossomos/metabolismo , Regiões 5' não Traduzidas , Fator 4 Ativador da Transcrição/metabolismo , Proteínas de Ciclo Celular/genética , Códon , Fator de Iniciação 2 em Eucariotos/genética , Fator de Iniciação 2 em Eucariotos/metabolismo , Fatores de Iniciação em Eucariotos/genética , Regulação da Expressão Gênica , Células HeLa , Humanos , Neoplasias/genética , Proteínas Oncogênicas/genética , Oncogenes , Fases de Leitura Aberta , RNA Mensageiro , RNA de Transferência/genética , RNA de Transferência/metabolismo , Subunidades Ribossômicas Menores de Eucariotos/genética , Subunidades Ribossômicas Menores de Eucariotos/metabolismo , Ribossomos/genéticaRESUMO
Regulation of mitosis secures cellular integrity and its failure critically contributes to the development, maintenance, and treatment resistance of cancer. In yeast, the dual phosphatase Cdc14 controls mitotic progression by antagonizing Cdk1-mediated protein phosphorylation. By contrast, specific mitotic functions of the mammalian Cdc14 orthologue CDC14B have remained largely elusive. Here, we find that CDC14B antagonizes CDK1-mediated activating mitotic phosphorylation of the deubiquitinase USP9X at serine residue 2563, which we show to be essential for USP9X to mediate mitotic survival. Starting from an unbiased proteome-wide screening approach, we specify Wilms' tumor protein 1 (WT1) as the relevant substrate that becomes deubiquitylated and stabilized by serine 2563-phosphorylated USP9X in mitosis. We further demonstrate that WT1 functions as a mitotic transcription factor and specify CXCL8/IL-8 as a target gene of WT1 that conveys mitotic survival. Together, we describe a ubiquitin-dependent signaling pathway that directs a mitosis-specific transcription program to regulate mitotic survival.
Assuntos
Proteína Quinase CDC2/antagonistas & inibidores , Fosfatases de Especificidade Dupla/antagonistas & inibidores , Mitose/fisiologia , Ubiquitina Tiolesterase/efeitos dos fármacos , Ubiquitina Tiolesterase/metabolismo , Proteínas WT1/metabolismo , Células A549 , Apoptose , Técnicas de Silenciamento de Genes , Células HEK293 , Células HeLa , Humanos , Interleucina-8/metabolismo , Fosforilação , Fatores de Transcrição , Ubiquitina Tiolesterase/genética , Proteínas WT1/genéticaRESUMO
The non-canonical initiation factors DENR and MCTS1 have been linked to cancer and autism. We recently showed in Drosophila that DENR and MCTS1 regulate translation re-initiation on transcripts containing upstream Open Reading Frames (uORFs) with strong Kozak sequences (stuORFs). Due to the medical relevance of DENR and MCTS1, it is worthwhile identifying the transcripts in human cells that depend on DENR and MCTS1 for their translation. We show here that in humans, as in Drosophila, transcripts with short stuORFs require DENR and MCTS1 for their optimal expression. In contrast to Drosophila, however, the dependence on stuORF length in human cells is very strong, so that only transcripts with very short stuORFs coding for 1 amino acid are dependent on DENR and MCTS1. This identifies circa 100 genes as putative DENR and MCTS1 translational targets. These genes are enriched for neuronal genes and G protein-coupled receptors. The identification of DENR and MCTS1 target transcripts will serve as a basis for future studies aimed at understanding the mechanistic involvement of DENR and MCTS1 in cancer and autism.
