MAPK-interacting kinase 2 (MNK2) regulates adipocyte metabolism independently of its catalytic activity

The mitogen-activated protein kinase (MAPK)-interacting kinases (MNKs) are serine/threonine protein kinases that are activated by the ERK1/2 (extracellular-regulated kinase) and p38α/β MAPK pathways. The MNKs have previously been implicated in metabolic disease and shown to mediate diet-induced obesity. In particular, knockout of MNK2 in mice protects from the weight gain induced by a high-fat diet. These and other data suggest that MNK2 regulates expansion of adipose tissue, a stable, long-term energy reserve that plays an important role in regulating whole-body energy homeostasis. Using the well-established mouse 3T3-L1 in vitro model of adipogenesis, the role of the MNKs in adipocyte differentiation and lipid storage was investigated. Inhibition of MNK activity using specific inhibitors failed to impair adipogenesis or lipid accumulation, suggesting that MNK activity is not required for adipocyte differentiation and does not regulate lipid storage. However, small-interfering RNA (siRNA) knockdown of MNK2 did reduce lipid accumulation and regulated the levels of two major lipogenic transcriptional regulators, ChREBP (carbohydrate response-element binding protein) and LPIN1 (Lipin-1). These factors are responsible for controlling expression of genes for proteins involved in de novo lipogenesis and triglyceride synthesis. Knockdown of MNK2 also increased the expression of hormone-sensitive lipase which catalyses the breakdown of triglyceride. These findings identify MNK2 as a regulator of adipocyte metabolism, independently of its catalytic activity, and reveal some of the mechanisms by which MNK2 drives adipose tissue expansion. The development of an MNK2-targeted therapy may therefore be a useful intervention for reducing weight caused by excessive nutrient intake.James E. Merrett, Jianling Xie, Peter J. Psaltis, and Christopher G. Prou

cAMP inhibits translation by inducing Ca2+/calmodulin-independent elongation factor 2 kinase activity in IPC-81 cells

AbstractTreatment of IPC-81 cells led to inhibition of protein synthesis, which was accompanied by an increase in the average size of polysomes and a decreased rate of elongation, indicating that it involved inhibition of peptide chain elongation. This inhibition was also associated with increased phosphorylation of elongation factor eEF2 (which inhibits its activity) and enhanced Ca2+/calmodulin-independent activity of eEF2 kinase. Previous work has shown that phosphorylation of eEF2 kinase by cAMP-dependent protein kinase (cAPK) in vitro induces such activator-independent activity, and the present data show that such a mechanism can occur in intact cells to link physiological levels of cAPK activation with inhibition of protein synthesis

Eukaryotic Elongation Factor 2 Kinase Activity Is Required for the Phenotypes of the Rpl24Bst Mouse

John R.P.Knight, Christopher G.Proud, Giovanna Mallucci, Tobiasvon der Haar, C. Mark Smales, Anne E.Willis, Owen J.Sanso

Deletion of Rptor in preosteoblasts reveals a role for the mammalian target of rapamycin complex 1 (mTORC1) complex in dietary-induced changes to bone mass and glucose homeostasis in female mice

The mammalian target of rapamycin complex 1 (mTORC1) complex is the major nutrient sensor in mammalian cells that responds to amino acids, energy levels, growth factors, and hormones, such as insulin, to control anabolic and catabolic processes. We have recently shown that suppression of the mTORC1 complex in bone-forming osteoblasts (OBs) improved glucose handling in male mice fed a normal or obesogenic diet. Mechanistically, this occurs, at least in part, by increasing OB insulin sensitivity leading to upregula- tion of glucose uptake and glycolysis. Given previously reported sex-dependent differences observed upon antagonism of mTORC1 signaling, we investigated the metabolic and skeletal effects of genetic inactivation of preosteoblastic-mTORC1 in female mice. Eight- week-old control diet (CD)-fed Rptor ob −/− mice had a low bone mass with a significant reduction in trabecular bone volume and trabecular number, reduced cortical bone thickness, and increased marrow adiposity. Despite no changes in body composition, CD-fed Rptor ob −/− mice exhibited significant lower fasting insulin and glucose levels and increased insulin sensitivity. Upon high-fat diet (HFD) feeding, Rptor ob −/− mice were resistant to a diet-induced increase in whole-body and total fat mass and protected from the development of diet-induced insulin resistance. Notably, although 12 weeks of HFD increased marrow adiposity, with minimal changes in both trabecular and cortical bone in the female control mice, marrow adiposity was significantly reduced in HFD-fed Rptor ob −/− compared to both HFD-fed control and CD-fed Rptor ob −/− mice. Collectively, our results demonstrate that mTORC1 func- tion in preosteoblasts is crucial for skeletal development and skeletal regulation of glucose homeostasis in both male and female mice. Importantly, loss of mTORC1 function in OBs results in metabolic and physiological adaptations that mirror a caloric restriction phenotype (under CD) and protects against HFD-induced obesity, associated insulin resistance, and marrow adiposity expansion. These results highlight the critical contribution of the skeleton in the regulation of whole-body energy homeostasisPawanrat Tangseefa, Sally K. Martin, Agnieszka Arthur, Vasilios Panagopoulos, Amanda J. Page, Gary A. Wittert ... et al

eEF2K enhances expression of PD-L1 by promoting the translation of its mRNA

Emerging advances in cancer therapy have transformed the landscape towards cancer immunotherapy regimens. Recent discoveries have resulted in the development of clinical immune checkpoint inhibitors that are 'game-changers' for cancer immunotherapy. Here we show that eEF2K, an atypical protein kinase that negatively modulates the elongation stage of protein synthesis, promotes the synthesis of PD-L1, an immune checkpoint protein which helps cancer cells to escape from immunosurveillance. Ablation of eEF2K in prostate and lung cancer cells markedly reduced the expression levels of the PD-L1 protein. We show that eEF2K promotes the association of PD-L1 mRNAs with translationally active polyribosomes and that translation of the PD-L1 mRNA is regulated by a uORF (upstream open reading-frame) within its 5'-UTR (5'-untranslated region) which starts with a non-canonical CUG as the initiation codon. This inhibitory effect is attenuated by eEF2K thereby allowing higher levels of translation of the PD-L1 coding region and enhanced expression of the PD-L1 protein. Moreover, eEF2K-depleted cancer cells are more vulnerable to immune attack by natural killer cells. Therefore, control of translation elongation can modulate the translation of this specific mRNA, one which contains an uORF that starts with CUG, and perhaps others that contain a similar feature. Taken together, our data reveal that eEF2K regulates PD-L1 expression at the level of the translation of its mRNA by virtue of a uORF in its 5'-region. This, and other roles of eEF2K in cancer cell biology (e.g., in cell survival and migration), may be exploited for the design of future therapeutic strategies.Yu Wu, Jianling Xie, Xin Jin, Roman V. Lenchine, Xuemin Wang, Danielle M. Fang ... et al

Reducing the environmental impact of surgery on a global scale: systematic review and co-prioritization with healthcare workers in 132 countries

Background Healthcare cannot achieve net-zero carbon without addressing operating theatres. The aim of this study was to prioritize feasible interventions to reduce the environmental impact of operating theatres. Methods This study adopted a four-phase Delphi consensus co-prioritization methodology. In phase 1, a systematic review of published interventions and global consultation of perioperative healthcare professionals were used to longlist interventions. In phase 2, iterative thematic analysis consolidated comparable interventions into a shortlist. In phase 3, the shortlist was co-prioritized based on patient and clinician views on acceptability, feasibility, and safety. In phase 4, ranked lists of interventions were presented by their relevance to high-income countries and low–middle-income countries. Results In phase 1, 43 interventions were identified, which had low uptake in practice according to 3042 professionals globally. In phase 2, a shortlist of 15 intervention domains was generated. In phase 3, interventions were deemed acceptable for more than 90 per cent of patients except for reducing general anaesthesia (84 per cent) and re-sterilization of ‘single-use’ consumables (86 per cent). In phase 4, the top three shortlisted interventions for high-income countries were: introducing recycling; reducing use of anaesthetic gases; and appropriate clinical waste processing. In phase 4, the top three shortlisted interventions for low–middle-income countries were: introducing reusable surgical devices; reducing use of consumables; and reducing the use of general anaesthesia. Conclusion This is a step toward environmentally sustainable operating environments with actionable interventions applicable to both high– and low–middle–income countries

Ras, PI3-kinase and mTOR signaling in cardiac hypertrophy

Cardiac hypertrophy involves increased mass (growth) of the heart and a cardinal feature of this condition is increased rates of protein synthesis. Several signaling pathways have been implicated in cardiac hypertrophy including the phosphatidylinositol 3-kinase (PI3K) and Ras/Raf/MEK/Erk pathways. PI3K lies upstream of the mammalian target of rapamycin (mTOR), an important positive regulator of protein synthesis and cell growth. However, recent data suggest that, in response to certain hypertrophic agents, signaling via Ras and MEK/Erk, as well as mTOR, is required for activation of protein synthesis, indicating new connections between these key signaling pathways

Protein synthesis in health and disease: editorial

The process of protein synthesis (mRNA translation) is essential for the growth and survival of all living organisms. Protein synthesis places heavy demands upon the cell—it requires a supply of amino acids as precursors and a great deal of energy. For these reasons, overall protein synthesis is under tight control. Furthermore, the translation of specific mRNAs is also subject to sophisticated control mechanisms, allowing the cell to modulate the production of certain proteins. Regulating translation rather than earlier stages in gene expression (transcription, splicing) confers important advantages—especially rapidity (by avoiding the requirement for de novo transcription and mRNA processing) and spatial control, e.g., in early development. Great strides have now been made in understanding how protein synthesis works and the functions and structures of the components involved.The chapters in this volume broadly concern two different areas of translational control. The first four primarily concern the regulation of the protein factors involved in the process of translation. This regulation generally involves alterations in their states of phosphorylation which influence their activity and interactions. The chapter by Proud describes the control of eukaryotic initiation factor 2. Its control has been intensively studied as it was a very early example of regulation of translation by protein phosphorylation. It is a target for four mammalian protein kinases which play important roles in regulating translation in response to diverse stress conditions. Recent data, using knock-in or knock-out mouse models, have clearly shown the importance of the kinases for normal physiology. It is also now clear that inherited mutations either in one of these kinases or in an ancillary factor for eIF2 (eIF2B) can lead to severe human diseases, as also discussed by Proud.Clemens discusses the mechanisms by which viruses seek to subvert the cellular protein synthesis machinery to favour expression of their own genetic information and the ways in which host cells strive to prevent this. A key player here is an eIF2 kinase termed PKR. Many of the mechanisms are also relevant in uninfected cells in response to stresses or early in apoptosis.As precursors for protein synthesis, amino acids exert important regulatory effects on the translational machinery. Some are exerted through eIF2, others through a key signaling pathway involving a protein termed the mammalian target of rapamycin, mTOR. Kimball focuses on recent studies that employ intact animals, rather than isolated cells, to explore the roles of amino acids in regulating translation factor activity.The mTOR pathway is currently the focus of intense attention. This reflects both its key roles in cellular and organismal physiology, and the links between this pathway and a range of human diseases including certain types of cancer and a condition termed tuberous sclerosis. Tee and Blenis discuss how studies on tuberous sclerosis have led to an improved understanding of mTOR signaling, and the roles of mTOR in health and disease.The second group of articles concern features of mRNA molecules themselves that are important for the control of their translation. In many cases, these features lie in the 5?- or 3?-untranslated regions (UTRs) of the message. Pickering and Willis review the roles of elements in the 5?-UTRs of certain mRNAs in mediating or controlling their translation. These include internal ribosome entry sites, which were initially found in viral mRNAs and which allow these mRNAs to be efficiently translated in the host cell.The articles by de Moor and colleagues and by Espel concern features found in the 3?-UTRs of specific mRNAs. The first chapter discusses 3?-UTR elements that are important in controlling the translation of specific mRNAs during early development and cell differentiation and during the cell cycle. The resulting translational control processes play critical roles in a diverse array of biological processes. A range of such regulatory elements have been identified, and these work by interacting with specific RNA-binding proteins. This is also true of the distinct 3?-UTR elements described by Espel: these AU-rich elements (AREs) play important roles in regulating the stability and translation of mRNAs encoding pro-inflammatory proteins and tumour-related proteins. As discussed by Espel, particular attention has focused on the role of AREs and ARE-binding proteins in inflammation, and on the signaling pathways involved.As the reader will appreciate from this set of articles, it is now abundantly clear that translational control is crucial for normal development and growth, and that dysregulation of these mechanisms leads to a range of serious human diseases.<br/

Regulation of protein synthesis by insulin

Insulin rapidly activates protein synthesis by activating components of the translational machinery including eIFs (eukaryotic initiation factors) and eEFs (eukaryotic elongation factors). In the long term, insulin also increases the cellular content of ribosomes to augment the capacity for protein synthesis. The rapid activation of protein synthesis by insulin is mediated primarily through phosphoinositide 3-kinase. This involves the activation of PKB (protein kinase B). In one case, PKB acts to phosphorylate and inactivate glycogen synthase kinase 3, which in turn phosphorylates and inhibits eIF2B. Insulin elicits the dephosphorylation and activation of eIF2B. Since eIF2B is required for recycling of eIF2, a factor required for all cytoplasmic translation initiation events, this will contribute to overall activation of protein synthesis. PKB also phosphorylates the TSC1 (tuberous sclerosis complex 1)–TSC2 complex to relieve its inhibitory action on the mTOR (mammalian target of rapamycin). Inhibition of mTOR by rapamycin markedly impairs insulin-activated protein synthesis. mTOR controls translation initiation and elongation. The cap-binding factor eIF4E can be sequestered in inactive complexes by 4E-BP1 (eIF4E-binding protein 1). Insulin elicits phosphorylation of 4E-BP1 and its release from eIF4E, allowing eIF4E to form initiation factor complexes. Insulin induces dephosphorylation and activation of eEF2 to accelerate elongation. Both effects are blocked by rapamycin. Insulin inactivates eEF2 kinase by increasing its phosphorylation at several mTOR-regulated sites. Insulin also stimulates synthesis of ribosomal proteins by promoting recruitment of their mRNAs into polyribosomes. This is inhibited by rapamycin. Several key questions remain about, for example, the mechanisms by which mTOR controls 4E-BP1 and eEF2 kinase and the control of ribosomal protein translation

mTOR-mediated regulation of translation factors by amino acids

The mammalian-target-of-rapamycin (mTOR) is a multidomain protein that is important in regulating several components of the translational machinery. mTOR signalling is stimulated by hormones (e.g., insulin) and by amino acids. Our recent data suggest that TOR signalling responds to intracellular amino acids rather than to external amino acid levels. The translational repressor eukaryotic initiation factor 4E-binding protein 1 (4E-BP1) is regulated through mTOR and undergoes phosphorylation at multiple sites, which affects its function. It contains two regulatory motifs: the C-terminal TOS motif interacts with the mTOR-binding partner, raptor, and mediates phosphorylation of specific sites in 4E-BP1. However, the N-terminal RAIP motif affects a larger range of mTOR-regulated sites. Since this motif does not bind raptor, mTOR must signal to 4E-BP1 via additional mechanisms that are independent of raptor. The kinase that phosphorylates and inhibits elongation factor 2 (eEF2 kinase) is inactivated by insulin via mTOR. Insulin decreases the ability of eEF2 kinase to bind calmodulin, its essential activator, and this effect requires mTOR signalling and a novel phosphorylation site in eEF2 kinase, Ser78. Ser78 is not phosphorylated by known components of the mTOR pathway implying the existence of novel mTOR-regulated kinases that control eEF2 kinase