scholarly journals LARP1 is a major phosphorylation substrate of mTORC1

2018 ◽  
Author(s):  
Bruno D. Fonseca ◽  
Jian-Jun Jia ◽  
Anne K. Hollensen ◽  
Roberta Pointet ◽  
Huy-Dung Hoang ◽  
...  
Keyword(s):  
Ribosome Biogenesis ◽  
Critical Role ◽  
Mammalian Target Of Rapamycin ◽  
Mrna Translation ◽  
Cellular Functions ◽  
Multiple Substrates ◽  
Eif4f Complex ◽  
Mtorc1 Pathway ◽  
Terminal Oligopyrimidine

AbstractThe mammalian target of rapamycin complex 1 (mTORC1) controls critical cellular functions such as protein synthesis, lipid metabolism, protein turnover and ribosome biogenesis through the phosphorylation of multiple substrates. In this study, we examined the phosphorylation of a recently identified target of mTORC1: La-related protein 1 (LARP1), a member of the LARP superfamily. Previously, we and others have shown that LARP1 plays an important role in repressing TOP mRNA translation downstream of mTORC1. LARP1 binds the 7-methylguanosine triphosphate (m7Gppp) cap moiety and the adjacent 5’terminal oligopyrimidine (5’TOP) motif of TOP mRNAs, thus impeding the assembly of the eIF4F complex on these transcripts. mTORC1 plays a critical role in the control of TOP mRNA translation via LARP1 but the precise mechanism by which this occurs is incompletely understood. The data described herein help to elucidate this process. Specifically, it show that: (i) mTORC1 interacts with LARP1, but not other LARP superfamily members, via the C-terminal region that comprises the DM15 domain, (ii) mTORC1 pathway controls the phosphorylation of multiple (up to 26) serine and threonine residues on LARP1 in vivo, (iii) mTORC1 regulates the binding of LARP1 to TOP mRNAs and (iv) phosphorylation of S689 by mTORC1 is particularly important for the association of the DM15 domain of LARP1 with the 5’UTR of RPS6 TOP mRNA. These data reveal LARP1 as a major substrate of mTORC1.

scholarly journals mTORC1-Activated S6K1 Phosphorylates Rictor on Threonine 1135 and Regulates mTORC2 Signaling

2009 ◽  
Vol 30 (4) ◽  
pp. 908-921 ◽  
Author(s):  
Louis-Andre Julien ◽  
Audrey Carriere ◽  
Julie Moreau ◽  
Philippe P. Roux
Keyword(s):  
Ribosome Biogenesis ◽  
Cellular Localization ◽  
Glycogen Synthase ◽  
Mammalian Target Of Rapamycin ◽  
Mrna Translation ◽  
Growth Factor Signaling ◽  
Mtorc1 Signaling ◽  
Ribosomal S6 Kinase

ABSTRACT The mammalian target of rapamycin (mTOR) is a conserved Ser/Thr kinase that forms two functionally distinct complexes important for nutrient and growth factor signaling. While mTOR complex 1 (mTORC1) regulates mRNA translation and ribosome biogenesis, mTORC2 plays an important role in the phosphorylation and subsequent activation of Akt. Interestingly, mTORC1 negatively regulates Akt activation, but whether mTORC1 signaling directly targets mTORC2 remains unknown. Here we show that growth factors promote the phosphorylation of Rictor (rapamycin-insensitive companion of mTOR), an essential subunit of mTORC2. We found that Rictor phosphorylation requires mTORC1 activity and, more specifically, the p70 ribosomal S6 kinase 1 (S6K1). We identified several phosphorylation sites in Rictor and found that Thr1135 is directly phosphorylated by S6K1 in vitro and in vivo, in a rapamycin-sensitive manner. Phosphorylation of Rictor on Thr1135 did not affect mTORC2 assembly, kinase activity, or cellular localization. However, cells expressing a Rictor T1135A mutant were found to have increased mTORC2-dependent phosphorylation of Akt. In addition, phosphorylation of the Akt substrates FoxO1/3a and glycogen synthase kinase 3α/β (GSK3α/β) was found to be increased in these cells, indicating that S6K1-mediated phosphorylation of Rictor inhibits mTORC2 and Akt signaling. Together, our results uncover a new regulatory link between the two mTOR complexes, whereby Rictor integrates mTORC1-dependent signaling.

scholarly journals A critical role for Apc in hematopoietic stem and progenitor cell survival

2008 ◽  
Vol 205 (9) ◽  
pp. 2163-2175 ◽  
Author(s):  
Zhijian Qian ◽  
Lina Chen ◽  
Anthony A. Fernald ◽  
Bart O. Williams ◽  
Michelle M. Le Beau
Keyword(s):  
Chromosome Segregation ◽  
Bone Marrow Failure ◽  
Critical Role ◽  
Cellular Functions ◽  
Erythroid Progenitor ◽  
Hematopoietic Stem ◽  
Myeloid Progenitor ◽  
Cell Cycle Entry ◽  
Genes Encoding

The adenomatous polyposis coli (Apc) tumor suppressor is involved in the initiation and progression of colorectal cancer via regulation of the Wnt signaling cascade. In addition, Apc plays an important role in multiple cellular functions, including cell migration and adhesion, spindle assembly, and chromosome segregation. However, its role during adult hematopoiesis is unknown. We show that conditional inactivation of Apc in vivo dramatically increases apoptosis and enhances cell cycle entry of hematopoietic stem cells (HSCs)/ hematopoietic progenitor cells (HPCs), leading to their rapid disappearance and bone marrow failure. The defect in HSCs/HPCs caused by Apc ablation is cell autonomous. In addition, we found that loss of Apc leads to exhaustion of the myeloid progenitor pool (common myeloid progenitor, granulocyte-monocyte progenitor, and megakaryocyte-erythroid progenitor), as well as the lymphoid-primed multipotent progenitor pool. Down-regulation of the genes encoding Cdkn1a, Cdkn1b, and Mcl1 occurs after acute Apc excision in candidate HSC populations. Together, our data demonstrate that Apc is essential for HSC and HPC maintenance and survival.

scholarly journals Chemokine-biased robust self-organizing polarization of migrating cells in vivo

2019 ◽  
Author(s):  
Adan Olguin-Olguin ◽  
Anne Aalto ◽  
Benoît Maugis ◽  
Michal Reichman-Fried ◽  
Erez Raz
Keyword(s):  
Primordial Germ Cells ◽  
Critical Role ◽  
Cell Polarization ◽  
Cellular Functions ◽  
Motile Cells ◽  
Cell Front ◽  
Specific Proteins ◽  
Migrating Cells

The mechanisms facilitating the establishment of front-rear polarity in migrating cells are not fully understood, in particular in the context of bleb-driven directional migration. To gain further insight into this issue we utilized the migration of zebrafish primordial germ cells (PGCs) as an in vivo model. We followed the molecular and morphological cascade that converts apolar cells into polarized bleb-forming motile cells and analyzed the cross dependency among the different cellular functions we identified. Our results underline the critical role of antagonistic interactions between the front and the rear, in particular the role of biophysical processes including formation of barriers and transport of specific proteins to the back of the cell. These interactions direct the formation of blebs to a specific part of the cell that is specified as the cell front. In this way, spontaneous cell polarization facilitates non-directional cell motility and when biased by chemokine signals leads to migration towards specific locations.

scholarly journals The Microtubule Severing Protein Katanin Regulates Proliferation of Neuronal Progenitors in Embryonic and Adult Neurogenesis

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Franco L. Lombino ◽  
Mary Muhia ◽  
Jeffrey Lopez-Rojas ◽  
Monika S. Brill ◽  
Edda Thies ◽  
...  
Keyword(s):  
Adult Neurogenesis ◽  
Critical Role ◽  
Mammalian Brain ◽  
Cellular Functions ◽  
Microtubule Severing ◽  
Neuronal Progenitor ◽  
Neuronal Progenitors ◽  
Adult Hippocampus

Abstract Microtubule severing regulates cytoskeletal rearrangement underlying various cellular functions. Katanin, a heterodimer, consisting of catalytic (p60) and regulatory (p80) subunits severs dynamic microtubules to modulate several stages of cell division. The role of p60 katanin in the mammalian brain with respect to embryonic and adult neurogenesis is poorly understood. Here, we generated a Katna1 knockout mouse and found that consistent with a critical role of katanin in mitosis, constitutive homozygous Katna1 depletion is lethal. Katanin p60 haploinsufficiency induced an accumulation of neuronal progenitors in the subventricular zone during corticogenesis, and impaired their proliferation in the adult hippocampus dentate gyrus (DG) subgranular zone. This did not compromise DG plasticity or spatial and contextual learning and memory tasks employed in our study, consistent with the interpretation that adult neurogenesis may be associated with selective forms of hippocampal-dependent cognitive processes. Our data identify a critical role for the microtubule-severing protein katanin p60 in regulating neuronal progenitor proliferation in vivo during embryonic development and adult neurogenesis.

scholarly journals S6K1−/−/S6K2−/− Mice Exhibit Perinatal Lethality and Rapamycin-Sensitive 5′-Terminal Oligopyrimidine mRNA Translation and Reveal a Mitogen-Activated Protein Kinase-Dependent S6 Kinase Pathway

2004 ◽  
Vol 24 (8) ◽  
pp. 3112-3124 ◽  
Author(s):  
Mario Pende ◽  
Sung Hee Um ◽  
Virginie Mieulet ◽  
Melanie Sticker ◽  
Valerie L. Goss ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Cell Cycle Progression ◽  
Mammalian Target Of Rapamycin ◽  
Mrna Translation ◽  
Mitogen Activated Protein Kinase ◽  
Dependent Manner ◽  
Wild Type ◽  
Mitogen Activated Protein ◽  
Terminal Oligopyrimidine ◽  
Perinatal Lethality

ABSTRACT Activation of 40S ribosomal protein S6 kinases (S6Ks) is mediated by anabolic signals triggered by hormones, growth factors, and nutrients. Stimulation by any of these agents is inhibited by the bacterial macrolide rapamycin, which binds to and inactivates the mammalian target of rapamycin, an S6K kinase. In mammals, two genes encoding homologous S6Ks, S6K1 and S6K2, have been identified. Here we show that mice deficient for S6K1 or S6K2 are born at the expected Mendelian ratio. Compared to wild-type mice, S6K1−/− mice are significantly smaller, whereas S6K2 −/− mice tend to be slightly larger. However, mice lacking both genes showed a sharp reduction in viability due to perinatal lethality. Analysis of S6 phosphorylation in the cytoplasm and nucleoli of cells derived from the distinct S6K genotypes suggests that both kinases are required for full S6 phosphorylation but that S6K2 may be more prevalent in contributing to this response. Despite the impairment of S6 phosphorylation in cells from S6K1 −/−/S6K2 −/− mice, cell cycle progression and the translation of 5′-terminal oligopyrimidine mRNAs were still modulated by mitogens in a rapamycin-dependent manner. Thus, the absence of S6K1 and S6K2 profoundly impairs animal viability but does not seem to affect the proliferative responses of these cell types. Unexpectedly, in S6K1 −/−/S6K2 −/− cells, S6 phosphorylation persisted at serines 235 and 236, the first two sites phosphorylated in response to mitogens. In these cells, as well as in rapamycin-treated wild-type, S6K1 −/−, and S6K2 −/− cells, this step was catalyzed by a mitogen-activated protein kinase (MAPK)-dependent kinase, most likely p90rsk. These data reveal a redundancy between the S6K and the MAPK pathways in mediating early S6 phosphorylation in response to mitogens.

scholarly journals Phosphorylation of Eukaryotic Translation Initiation Factor 2α Coordinates rRNA Transcription and Translation Inhibition during Endoplasmic Reticulum Stress

2009 ◽  
Vol 29 (15) ◽  
pp. 4295-4307 ◽  
Author(s):  
Jenny B. DuRose ◽  
Donalyn Scheuner ◽  
Randal J. Kaufman ◽  
Lawrence I. Rothblum ◽  
Maho Niwa
Keyword(s):  
Endoplasmic Reticulum ◽  
Ribosome Biogenesis ◽  
Critical Role ◽  
Mammalian Target Of Rapamycin ◽  
Protein Translation ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Rrna Synthesis ◽  
Cell Physiology ◽  
Rrna Transcription

ABSTRACT The endoplasmic reticulum (ER) is the major cellular compartment where folding and maturation of secretory and membrane proteins take place. When protein folding needs exceed the capacity of the ER, the unfolded protein response (UPR) pathway modulates gene expression and downregulates protein translation to restore homeostasis. Here, we report that the UPR downregulates the synthesis of rRNA by inactivation of the RNA polymerase I basal transcription factor RRN3/TIF-IA. Inhibition of rRNA synthesis does not appear to involve the well-characterized mTOR (mammalian target of rapamycin) pathway; instead, PERK-dependent phosphorylation of eIF2α plays a critical role in the inactivation of RRN3/TIF-IA. Downregulation of rRNA transcription occurs simultaneously or slightly prior to eIF2α phosphorylation-induced translation repression. Since rRNA is the most abundant RNA species, constituting ∼90% of total cellular RNA, its downregulation exerts a significant impact on cell physiology. Our study demonstrates the first link between regulation of translation and rRNA synthesis with phosphorylation of eIF2α, suggesting that this pathway may be broadly utilized by stresses that activate eIF2α kinases in order to coordinately regulate translation and ribosome biogenesis during cellular stress.

scholarly journals The sequence of the 5' end of the U8 small nucleolar RNA is critical for 5.8S and 28S rRNA maturation.

1997 ◽  
Vol 17 (7) ◽  
pp. 3702-3713 ◽  
Author(s):  
B A Peculis
Keyword(s):  
Ribosome Biogenesis ◽  
Critical Role ◽  
Functional Site ◽  
28S Rrna ◽  
Rrna Processing ◽  
Hybrid Molecules ◽  
Evolutionarily Conserved ◽  
Rrna Maturation ◽  
Base Pairing Interaction

Ribosome biogenesis in eucaryotes involves many small nucleolar ribonucleoprotein particles (snoRNP), a few of which are essential for processing pre-rRNA. Previously, U8 snoRNA was shown to play a critical role in pre-rRNA processing, being essential for accumulation of mature 28S and 5.8S rRNAs. Here, evidence which identifies a functional site of interaction on the U8 RNA is presented. RNAs with mutations, insertions, or deletions within the 5'-most 15 nucleotides of U8 do not function in pre-rRNA processing. In vivo competitions in Xenopus oocytes with 2'O-methyl oligoribonucleotides have confirmed this region as a functional site of a base-pairing interaction. Cross-species hybrid molecules of U8 RNA show that this region of the U8 snoRNP is necessary for processing of pre-rRNA but not sufficient to direct efficient cleavage of the pre-rRNA substrate; the structure or proteins comprising, or recruited by, the U8 snoRNP modulate the efficiency of cleavage. Intriguingly, these 15 nucleotides have the potential to base pair with the 5' end of 28S rRNA in a region where, in the mature ribosome, the 5' end of 28S interacts with the 3' end of 5.8S. The 28S-5.8S interaction is evolutionarily conserved and critical for pre-rRNA processing in Xenopus laevis. Taken together these data strongly suggest that the 5' end of U8 RNA has the potential to bind pre-rRNA and in so doing, may regulate or alter the pre-rRNA folding pathway. The rest of the U8 particle may then facilitate cleavage or recruitment of other factors which are essential for pre-rRNA processing.

A critical role for non-coding RNA GAS5 in growth arrest and rapamycin inhibition in human T-lymphocytes

2011 ◽  
Vol 39 (2) ◽  
pp. 482-486 ◽  
Author(s):  
Gwyn T. Williams ◽  
Mirna Mourtada-Maarabouni ◽  
Farzin Farzaneh
Keyword(s):  
T Lymphocytes ◽  
Critical Role ◽  
Mammalian Target Of Rapamycin ◽  
Growth Arrest ◽  
Normal Growth ◽  
Functional Expression ◽  
Expression Cloning ◽  
Human T Lymphocytes ◽  
Non Coding Rna ◽  
Terminal Oligopyrimidine

Non-coding RNA GAS5 (growth arrest-specific transcript 5) is a 5′-TOP (5′-terminal oligopyrimidine tract) RNA, whose translation, and consequently also stability, is controlled by the mTOR (mammalian target of rapamycin) pathway. GAS5 was identified by functional expression cloning and is necessary and sufficient for normal growth arrest in both leukaemic and untransformed human T-lymphocytes. GAS5 is also required for the inhibitory effects of rapamycin and its analogues on T-cells. The striking functional effects of GAS5 may be mediated through the snoRNAs (small nucleolar RNAs) encoded in its introns and/or through the unusual folding of the mRNA itself, which sequesters, and therefore inhibits, the glucocorticoid receptor.

scholarly journals An in vivo and in vitro assessment of TOR signaling cascade in rainbow trout (Oncorhynchus mykiss)

2008 ◽  
Vol 295 (1) ◽  
pp. R329-R335 ◽  
Author(s):  
Iban Seiliez ◽  
Jean-Charles Gabillard ◽  
Sandrine Skiba-Cassy ◽  
Daniel Garcia-Serrana ◽  
Joaquim Gutiérrez ◽  
...  
Keyword(s):  
Rainbow Trout ◽  
Amino Acid ◽  
Mammalian Target Of Rapamycin ◽  
Mrna Translation ◽  
Target Of Rapamycin ◽  
Nutritional Regulation ◽  
Tor Signaling ◽  
Meal Feeding

In mammals, feeding promotes protein accretion in skeletal muscle through a stimulation of the insulin- and amino acid- sensitive mammalian target of rapamycin (mTOR) signaling pathway, leading to the induction of mRNA translation. The purpose of the present study was to characterize both in vivo and in vitro the activation of several major kinases involved in the mTOR pathway in the muscle of the carnivorous rainbow trout. Our results showed that meal feeding enhanced the phosphorylation of the target of rapamycin (TOR), PKB, p70 S6 kinase, and eIF4E-binding protein-1, suggesting that the mechanisms involved in the regulation of mRNA translation are well conserved between lower and higher vertebrates. Our in vitro studies on primary culture of trout muscle cells indicate that insulin and amino acids regulate TOR signaling and thus may be involved in meal feeding effect in this species as in mammals. In conclusion, we report here for the first time in a fish species, the existence and the nutritional regulation of several major kinases involved in the TOR pathway, opening a new area of research on the molecular bases of amino acid utilization in teleosts.

scholarly journals mTORC1 promotes TOP mRNA translation through site-specific phosphorylation of LARP1

2021 ◽  
Author(s):  
Jian-Jun Jia ◽  
Roni M Lahr ◽  
Michael T Solgaard ◽  
Bruno J Moraes ◽  
Roberta Pointet ◽  
...  
Keyword(s):  
Rna Binding ◽  
Mrna Translation ◽  
Translation Control ◽  
Site Specific ◽  
Translation Repression ◽  
Eif4f Complex ◽  
Inhibitory Activities ◽  
Activation Status

Abstract LARP1 is a key repressor of TOP mRNA translation. It binds the m7Gppp cap moiety and the adjacent 5′TOP motif of TOP mRNAs, thus impeding the assembly of the eIF4F complex on these transcripts. mTORC1 controls TOP mRNA translation via LARP1, but the details of the mechanism are unclear. Herein we elucidate the mechanism by which mTORC1 controls LARP1’s translation repression activity. We demonstrate that mTORC1 phosphorylates LARP1 in vitro and in vivo, activities that are efficiently inhibited by rapamycin and torin1. We uncover 26 rapamycin-sensitive phospho-serine and -threonine residues on LARP1 that are distributed in 7 clusters. Our data show that phosphorylation of a cluster of residues located proximally to the m7Gppp cap-binding DM15 region is particularly sensitive to rapamycin and regulates both the RNA-binding and the translation inhibitory activities of LARP1. Our results unravel a new model of translation control in which the La module (LaMod) and DM15 region of LARP1, both of which can directly interact with TOP mRNA, are differentially regulated: the LaMod remains constitutively bound to PABP (irrespective of the activation status of mTORC1), while the C-terminal DM15 ‘pendular hook’ engages the TOP mRNA 5′-end to repress translation, but only in conditions of mTORC1 inhibition.

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