Régulation du métabolisme des ARNm par les voies de signalisation MAPK et mTOR

Il est à ce jour bien établi que la régulation de l’expression génique dépend en grande partie des évènements post-transcriptionnels et que la traduction des ARNm tient un rôle de premier plan dans ces processus. Elle est particulièrement importante pour définir le protéome, maintenir l’homéostasie et contrôler la croissance et la prolifération cellulaire. De nombreuses pathologies humaines telles que le cancer découlent de dérèglements de la synthèse protéique. Ceci souligne l’importance d’une meilleure compréhension des mécanismes moléculaires contribuant au contrôle de la traduction des ARNm. Le facteur d’initiation eIF4E est essentiel à la traduction et son activité est régulée par ses partenaires protéiques dont font partie les protéines 4E-BP et 4E-T. Les voies de signalisation PI3K/mTOR et MAPK qui sont fortement impliquées dans l’étiologie du cancer, contrôlent la traduction en modulant l’activité d’eIF4E via l’inhibition des protéines 4E-BP et la localisation de 4E-T. Afin d’améliorer notre compréhension des mécanismes régulant la traduction des ARNm, nous avons utilisé plusieurs approches. Tout d’abord, nous avons caractérisé les mécanismes par lesquels le complexe mTORC1 est activé en réponse aux facteurs de croissance et avons déterminé que la kinase RSK, en aval de la voie Ras/ERK, contrôle directement l’activité de mTORC1 en phosphorylant Raptor, la sous-unité régulatrice du complexe mTORC1. Par ailleurs, nous nous sommes intéressés au rôle joué par mTORC1 dans l’initiation de la traduction. Pour cela, nous avons réalisé un criblage protéomique dans le but d’identifier de nouveaux facteurs sous le contrôle de mTORC1 qui participent activement à la traduction. Ces travaux ont ainsi permis l’identification de la protéine de liaison à l’ARN LARP1 comme effecteur majeur de la traduction des ARNm et de la croissance cellulaire en aval de mTORC1. Finalement, notre étude de l’effet du stress oxydant dans la répression de la traduction nous a permis de montrer que la kinase JNK contrôle la localisation du répresseur 4E-T au sein des P-bodies, qui sont des granules cytoplasmiques concentrant des ARNm non traduits et des facteurs de la dégradation des ARNm. Nos travaux ont donc abouti à la découverte de mécanismes moléculaires cruciaux impliqués dans la régulation de la traduction des ARNm et de la synthèse protéique. Ces derniers étant largement impliqués dans la prolifération cellulaire et la croissance tumorale, nos recherches ouvrent sur un champ d’investigation plus large pour le développement de nouvelles molécules anti-cancéreuses.It is now well established that gene expression is predominantly regulated by post-transcriptional events and that mRNA translation plays an essential role in this process. Translation of mRNAs is especially important in defining the proteome, maintaining homeostasis and controlling cell growth and cell proliferation. Several human diseases such as cancer are associated with aberrant regulation of protein synthesis highlighting the need to better understand the molecular mechanisms contributing to translational control. The translation initiation factor eIF4E is a key component of the translational machinery whose activity is controlled by its partners, the 4E-BP and 4E-T proteins. The PI3K/mTOR and MAPK signaling pathways, which are strongly implicated in cancer etiology, control mRNA translation by modulating eIF4E activity through the inhibition of the 4E-BPs and the regulation of eIF4E localization by 4E-T. In order to better understand how mRNA translation is regulated we used several approaches. First, we characterized the mechanisms contributing to mTORC1 activation in response to growth factor. We found that the kinase RSK, that lies downstream of the Ras/ERK pathway, directly controls mTORC1 activity by phosphorylating Raptor, the regulatory sub-unit of the complex. This provides evidence of an additional mechanism by which MAPK pathway regulates mTORC1. We next performed a proteomic screen to identify novel mTOR-regulated factors that actively participate in translation. This approach led to the identification of several candidate proteins which included the RNA-binding protein LARP1 that we found to be a major effector of mTORC1-mediated mRNA translation, cell growth and proliferation. Finally we investigated the impact of oxidative stress on translation inhibition and found that the JNK kinase controls 4E-T localization in P-bodies that are cytoplasmic granules containing non-translating mRNAs and proteins from the mRNA decay and silencing machineries. Together this work provides important novel insights into the regulation of mRNA translation and protein synthesis that represent processes strongly connected to tumorigenesis and brings precious information on the mechanisms by which signaling pathways control cell growth and proliferation

Receptor tyrosine kinases: Characterisation, mechanism of action and therapeutic interests for bone cancers

Bone cancers are characterised by the development of tumour cells in bone sites, associated with a dysregulation of their environment. In the last two decades, numerous therapeutic strategies have been developed to target the cancer cells or tumour niche. As the crosstalk between these two entities is tightly controlled by the release of polypeptide mediators activating signalling pathways through several receptor tyrosine kinases (RTKs), RTK inhibitors have been designed. These inhibitors have shown exciting clinical impacts, such as imatinib mesylate, which has become a reference treatment for chronic myeloid leukaemia and gastrointestinal tumours. The present review gives an overview of the main molecular and functional characteristics of RTKs, and focuses on the clinical applications that are envisaged and already assessed for the treatment of bone sarcomas and bone metastases

Activation and Function of the MAPKs and Their Substrates, the MAPK-Activated Protein Kinases

Summary: The mitogen-activated protein kinases (MAPKs) regulate diverse cellular programs by relaying extracellular signals to intracellular responses. In mammals, there are more than a dozen MAPK enzymes that coordinately regulate cell proliferation, differentiation, motility, and survival. The best known are the conventional MAPKs, which include the extracellular signal-regulated kinases 1 and 2 (ERK1/2), c-Jun amino-terminal kinases 1 to 3 (JNK1 to -3), p38 (α, β, γ, and δ), and ERK5 families. There are additional, atypical MAPK enzymes, including ERK3/4, ERK7/8, and Nemo-like kinase (NLK), which have distinct regulation and functions. Together, the MAPKs regulate a large number of substrates, including members of a family of protein Ser/Thr kinases termed MAPK-activated protein kinases (MAPKAPKs). The MAPKAPKs are related enzymes that respond to extracellular stimulation through direct MAPK-dependent activation loop phosphorylation and kinase activation. There are five MAPKAPK subfamilies: the p90 ribosomal S6 kinase (RSK), the mitogen- and stress-activated kinase (MSK), the MAPK-interacting kinase (MNK), the MAPK-activated protein kinase 2/3 (MK2/3), and MK5 (also known as p38-regulated/activated protein kinase [PRAK]). These enzymes have diverse biological functions, including regulation of nucleosome and gene expression, mRNA stability and translation, and cell proliferation and survival. Here we review the mechanisms of MAPKAPK activation by the different MAPKs and discuss their physiological roles based on established substrates and recent discoveries

Extracellular Signal-Regulated Kinases 1 and 2 Phosphorylate Gab2 To Promote a Negative-Feedback Loop That Attenuates Phosphoinositide 3-Kinase/Akt Signaling.

The scaffolding adapter protein Gab2 (Grb2-associated binder) promotes cell proliferation, survival, and motility by engaging several signaling pathways downstream of growth factor and cytokine receptors. In particular, Gab2 plays essential roles in mast cells, as it is required for phosphoinositide 3-kinase (PI3K) activation in response to Kit and the high-affinity IgE receptor. While the positive role of Gab2 in PI3K signaling is well documented, very little is known about the mechanisms that attenuate its function. Here we show that Gab2 becomes phosphorylated on multiple proline-directed sites upon stimulation of the Ras/extracellular signal-regulated kinase (ERK) signaling pathway. We demonstrate that ERK1 and ERK2 interact with Gab2 via a novel docking motif, which is required for subsequent Gab2 phosphorylation in response to ERK1/2 activation. We identified four ERK1/2-dependent phosphorylation sites in Gab2 that prevent the recruitment of the p85 regulatory subunit of PI3K. Using bone marrow-derived mast cells to study Gab2-dependent signaling, we found that the inhibition of ERK1/2 activity promotes Akt signaling in response to Kit and the high-affinity IgE receptor. Together, our results indicate that ERK1/2 participates in a negative-feedback loop that attenuates PI3K/Akt signaling in response to various agonists

nanoCAGE reveals 5' UTR features that define specific modes of translation of functionally related MTOR-sensitive mRNAs

The diversity of MTOR-regulated mRNA translation remains unresolved. Whereas ribosome-profiling suggested that MTOR almost exclusively stimulates translation of the TOP (terminal oligopyrimidine motif) and TOP-like mRNAs, polysome-profiling indicated that MTOR also modulates translation of mRNAs without the 5' TOP motif (non-TOP mRNAs). We demonstrate that in ribosome-profiling studies, detection of MTOR-dependent changes in non-TOP mRNA translation was obscured by low sensitivity and methodology biases. Transcription start site profiling using nano-cap analysis of gene expression (nanoCAGE) revealed that not only do many MTOR-sensitive mRNAs lack the 5' TOP motif but that 5' UTR features distinguish two functionally and translationally distinct subsets of MTOR-sensitive mRNAs: (1) mRNAs with short 5' UTRs enriched for mitochondrial functions, which require EIF4E but are less EIF4A1-sensitive; and (2) long 5' UTR mRNAs encoding proliferation- and survival-promoting proteins, which are both EIF4E- and EIF4A1-sensitive. Selective inhibition of translation of mRNAs harboring long 5' UTRs via EIF4A1 suppression leads to sustained expression of proteins involved in respiration but concomitant loss of those protecting mitochondrial structural integrity, resulting in apoptosis. Conversely, simultaneous suppression of translation of both long and short 5' UTR mRNAs by MTOR inhibitors results in metabolic dormancy and a predominantly cytostatic effect. Thus, 5' UTR features define different modes of MTOR-sensitive translation of functionally distinct subsets of mRNAs, which may explain the diverse impact of MTOR and EIF4A inhibitors on neoplastic cells.P50 GM067041 - NIGMS NIH HHS; R01 GM073855 - NIGMS NIH HH

nanoCAGE reveals 5′ UTR features that define specific modes of translation of functionally related MTOR-sensitive mRNAs

The diversity of MTOR-regulated mRNA translation remains unresolved. Whereas ribosome-profiling suggested that MTOR almost exclusively stimulates translation of the TOP (terminal oligopyrimidine motif) and TOP-like mRNAs, polysome-profiling indicated that MTOR also modulates translation of mRNAs without the 5' TOP motif (non-TOP mRNAs). We demonstrate that in ribosome-profiling studies, detection of MTOR-dependent changes in non-TOP mRNA translation was obscured by low sensitivity and methodology biases. Transcription start site profiling using nano-cap analysis of gene expression (nanoCAGE) revealed that not only do many MTOR-sensitive mRNAs lack the 5' TOP motif but that 5' UTR features distinguish two functionally and translationally distinct subsets of MTOR-sensitive mRNAs: (1) mRNAs with short 5' UTRs enriched for mitochondrial functions, which require EIF4E but are less EIF4A1-sensitive; and (2) long 5' UTR mRNAs encoding proliferation- and survival-promoting proteins, which are both EIF4E- and EIF4A1-sensitive. Selective inhibition of translation of mRNAs harboring long 5' UTRs via EIF4A1 suppression leads to sustained expression of proteins involved in respiration but concomitant loss of those protecting mitochondrial structural integrity, resulting in apoptosis. Conversely, simultaneous suppression of translation of both long and short 5' UTR mRNAs by MTOR inhibitors results in metabolic dormancy and a predominantly cytostatic effect. Thus, 5' UTR features define different modes of MTOR-sensitive translation of functionally distinct subsets of mRNAs, which may explain the diverse impact of MTOR and EIF4A inhibitors on neoplastic cells.P50 GM067041 - NIGMS NIH HHS; R01 GM073855 - NIGMS NIH HH

mTORC1 and CK2 coordinate ternary and eIF4F complex assembly

International audienceTernary complex (TC) and eIF4F complex assembly are the two major rate-limiting steps in translation initiation regulated by eIF2α phosphorylation and the mTOR/4E-BP pathway, respectively. How TC and eIF4F assembly are coordinated, however, remains largely unknown. We show that mTOR suppresses translation of mRNAs activated under short-term stress wherein TC recycling is attenuated by eIF2α phosphorylation. During acute nutrient or growth factor stimulation, mTORC1 induces eIF2β phosphorylation and recruitment of NCK1 to eIF2, decreases eIF2α phosphorylation and bolsters TC recycling. Accordingly, eIF2β mediates the effect of mTORC1 on protein synthesis and proliferation. In addition, we demonstrate a formerly undocumented role for CK2 in regulation of translation initiation, whereby CK2 stimulates phosphorylation of eIF2β and simultaneously bolsters eIF4F complex assembly via the mTORC1/4E-BP pathway. These findings imply a previously unrecognized mode of translation regulation, whereby mTORC1 and CK2 coordinate TC and eIF4F complex assembly to stimulate cell proliferation