ATF4-Mediated Upregulation of REDD1 and Sestrin2 Suppresses mTORC1 Activity during Prolonged Leucine Deprivation

ABSTRACT Background The protein kinase target of rapamycin (mTOR) in complex 1 (mTORC1) is activated by amino acids and in turn upregulates anabolic processes. Under nutrient-deficient conditions, e.g., amino acid insufficiency, mTORC1 activity is suppressed and autophagy is activated. Intralysosomal amino acids generated by autophagy reactivate mTORC1. However, sustained mTORC1 activation during periods of nutrient insufficiency would likely be detrimental to cellular homeostasis. Thus, mechanisms must exist to prevent amino acids released by autophagy from reactivating the kinase. Objective The objective of the present study was to test whether mTORC1 activity is inhibited during prolonged leucine deprivation through ATF4-dependent upregulation of the mTORC1 suppressors regulated in development and DNA damage response 1 (REDD1) and Sestrin2. Methods Mice (8 wk old; C57Bl/6 × 129SvEV) were food deprived (FD) overnight and one-half were refed the next morning. Mouse embryo fibroblasts (MEFs) deficient in ATF4, REDD1, and/or Sestrin2 were deprived of leucine for 0–16 h. mTORC1 activity and ATF4, REDD1, and Sestrin2 expression were assessed in liver and cell lysates. Results Refeeding FD mice resulted in activation of mTORC1 in association with suppressed expression of both REDD1 and Sestrin2 in the liver. In cells in culture, mTORC1 exhibited a triphasic response to leucine deprivation, with an initial suppression followed by a transient reactivation from 2 to 4 h and a subsequent resuppression after 8 h. Resuppression occurred concomitantly with upregulated expression of ATF4, REDD1, and Sestrin2. However, in cells lacking ATF4, neither REDD1 nor Sestrin2 expression was upregulated by leucine deprivation, and resuppression of mTORC1 was absent. Moreover, in cells lacking either REDD1 or Sestrin2, mTORC1 resuppression was attenuated, and in cells lacking both proteins resuppression was further blunted. Conclusions The results suggest that leucine deprivation upregulates expression of both REDD1 and Sestrin2 in an ATF4-dependent manner, and that upregulated expression of both proteins is involved in resuppression of mTORC1 during prolonged leucine deprivation.

Download Full-text

In VivoAnalysis of Protein Kinase B (PKB)/Akt Regulation in DNA-PKcs-null Mice Reveals a Role for PKB/Akt in DNA Damage Response and Tumorigenesis

Journal of Biological Chemistry ◽

10.1074/jbc.m803053200 ◽

2008 ◽

Vol 283 (44) ◽

pp. 30025-30033 ◽

Cited By ~ 54

Author(s):

Banu Surucu ◽

Lana Bozulic ◽

Debby Hynx ◽

Arnaud Parcellier ◽

Brian A. Hemmings

Keyword(s):

Dna Damage ◽

Protein Kinase ◽

Dna Damage Response ◽

Protein Kinase B ◽

Damage Response

Download Full-text

HPF1 dynamically controls the PARP1/2 balance between initiating and elongating ADP-ribose modifications

Nature Communications ◽

10.1038/s41467-021-27043-8 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Marie-France Langelier ◽

Ramya Billur ◽

Aleksandr Sverzhinsky ◽

Ben E. Black ◽

John M. Pascal

Keyword(s):

Dna Damage ◽

Dna Damage Response ◽

Chain Elongation ◽

Dna Breaks ◽

Data Support ◽

Damage Response ◽

Hit And Run ◽

Dna Break ◽

Structural Insights ◽

In Cells

AbstractPARP1 and PARP2 produce poly(ADP-ribose) in response to DNA breaks. HPF1 regulates PARP1/2 catalytic output, most notably permitting serine modification with ADP-ribose. However, PARP1 is substantially more abundant in cells than HPF1, challenging whether HPF1 can pervasively modulate PARP1. Here, we show biochemically that HPF1 efficiently regulates PARP1/2 catalytic output at sub-stoichiometric ratios matching their relative cellular abundances. HPF1 rapidly associates/dissociates from multiple PARP1 molecules, initiating serine modification before modification initiates on glutamate/aspartate, and accelerating initiation to be more comparable to elongation reactions forming poly(ADP-ribose). This “hit and run” mechanism ensures HPF1 contributions to PARP1/2 during initiation do not persist and interfere with PAR chain elongation. We provide structural insights into HPF1/PARP1 assembled on a DNA break, and assess HPF1 impact on PARP1 retention on DNA. Our data support the prevalence of serine-ADP-ribose modification in cells and the efficiency of serine-ADP-ribose modification required for an acute DNA damage response.

Download Full-text

Beyond ATM: The protein kinase landscape of the DNA damage response

FEBS Letters ◽

10.1016/j.febslet.2011.05.013 ◽

2011 ◽

Vol 585 (11) ◽

pp. 1625-1639 ◽

Cited By ~ 131

Author(s):

Ariel Bensimon ◽

Ruedi Aebersold ◽

Yosef Shiloh

Keyword(s):

Dna Damage ◽

Protein Kinase ◽

Dna Damage Response ◽

Damage Response

Download Full-text

High Fidelity Deep Sequencing Reveals No Effect of ATM, ATR, and DNA-PK Cellular DNA Damage Response Pathways on Adenovirus Mutation Rate

Viruses ◽

10.3390/v11100938 ◽

2019 ◽

Vol 11 (10) ◽

pp. 938 ◽

Cited By ~ 1

Author(s):

Risso-Ballester ◽

Sanjuán

Keyword(s):

Dna Damage ◽

Protein Kinase ◽

Dna Damage Response ◽

Deep Sequencing ◽

Molecular Mechanisms ◽

Spontaneous Mutation ◽

High Fidelity ◽

Dna Viruses ◽

Damage Response ◽

Cellular Dna

Most DNA viruses exhibit relatively low rates of spontaneous mutation. However, the molecular mechanisms underlying DNA virus genetic stability remain unclear. In principle, mutation rates should not depend solely on polymerase fidelity, but also on factors such as DNA damage and repair efficiency. Most eukaryotic DNA viruses interact with the cellular DNA damage response (DDR), but the role of DDR pathways in preventing mutations in the virus has not been tested empirically. To address this goal, we serially transferred human adenovirus type 5 in cells in which the telangiectasia-mutated PI3K-related protein kinase (ATM), the ATM/Rad3-related (ATR) kinase, and the DNA-dependent protein kinase (DNA-PK) were chemically inactivated, as well as in control cells displaying normal DDR pathway functioning. High-fidelity deep sequencing of these viral populations revealed mutation frequencies in the order of one-millionth, with no detectable effect of the inactivation of DDR mediators ATM, ATR, and DNA-PK on adenovirus sequence variability. This suggests that these DDR pathways do not play a major role in determining adenovirus genetic diversity.

Download Full-text

Ribosomal protein L6 (RPL6) is recruited to DNA damage sites in a poly(ADP-ribose) polymerase–dependent manner and regulates the DNA damage response

Journal of Biological Chemistry ◽

10.1074/jbc.ra118.007009 ◽

2018 ◽

Vol 294 (8) ◽

pp. 2827-2838 ◽

Cited By ~ 10

Author(s):

Chuanzhen Yang ◽

Weicheng Zang ◽

Yapeng Ji ◽

Tingting Li ◽

Yongfeng Yang ◽

...

Keyword(s):

Dna Damage ◽

Ribosomal Protein ◽

Dna Damage Response ◽

Dependent Manner ◽

Damage Response

Download Full-text

Homeodomain-interacting protein kinase 2 regulates DNA damage response through interacting with heterochromatin protein 1γ

Oncogene ◽

10.1038/onc.2014.278 ◽

2014 ◽

Vol 34 (26) ◽

pp. 3463-3473 ◽

Cited By ~ 12

Author(s):

Y Akaike ◽

Y Kuwano ◽

K Nishida ◽

K Kurokawa ◽

K Kajita ◽

...

Keyword(s):

Dna Damage ◽

Protein Kinase ◽

Dna Damage Response ◽

Heterochromatin Protein ◽

Interacting Protein ◽

Damage Response

Download Full-text

Coordinated Chromatin-Association of Fanconi Anemia Network Proteins Requires Replication-Coupled DNA Damage Recognition.

Blood ◽

10.1182/blood.v104.11.723.723 ◽

2004 ◽

Vol 104 (11) ◽

pp. 723-723

Author(s):

Alexandra Sobeck ◽

Stacie Stone ◽

Bendert deGraaf ◽

Vincenzo Costanzo ◽

Johan deWinter ◽

...

Keyword(s):

Dna Damage ◽

Dna Replication ◽

Dna Damage Response ◽

Fanconi Anemia ◽

Bone Marrow Failure ◽

S Phase ◽

Dependent Manner ◽

Core Complex ◽

Damage Response ◽

Crosslinking Agents

Abstract Fanconi anemia (FA) is a genetic disorder characterized by hypersensitivity to DNA crosslinking agents and diverse clinical symptoms, including developmental anomalies, progressive bone marrow failure, and predisposition to leukemias and other cancers. FA is genetically heterogeneous, resulting from mutations in any of at least eleven different genes. The FA proteins function together in a pathway composed of a mulitprotein core complex that is required to trigger the DNA-damage dependent activation of the downstream FA protein, FANCD2. This activation is thought to be the key step in a DNA damage response that functionally links FA proteins to major breast cancer susceptibility proteins BRCA1 and BRCA2 (BRCA2 is FA gene FANCD1). The essential function of the FA proteins is unknown, but current models suggest that FA proteins function at the interface between cell cycle checkpoints, DNA repair and DNA replication, and are likely to play roles in the DNA damage response during S phase. To provide a platform for dissecting the key functional events during S-phase, we developed cell-free assays for FA proteins based on replicating extracts from Xenopus eggs. We identified the Xenopus homologs of human FANCD2 (xFANCD2) and several of the FA core complex proteins (xCCPs), and biochemically characterized these proteins in replicating cell-free extracts. We found that xCCPs and a modified isoform of xFANCD2 become associated with chromatin during normal and disrupted DNA replication. Blocking initiation of replication with geminin demonstrated that association of xCCPs and xFANCD2 with chromatin occurs in a strictly replication-dependent manner that is enhanced following DNA damage by crosslinking agents or by addition of aphidicolin, an inhibitor of replicative DNA polymerases. In addition, chromatin binding of xFANCD2, but not xBRCA2, is abrogated when xFANCA is quantitatively depleted from replicating extracts suggesting that xFANCA promotes the loading of xFANCD2 on chromatin. The chromatin-association of xFANCD2 and xCCPs is diminished in the presence of caffeine, an inhibitor of checkpoint kinases. Taken together, our data suggest a model in which the ordered loading of FA proteins on chromatin is required for processing a subset of DNA replication-blocking lesions that are resolved during late stages of replication.

Download Full-text

ATM regulates a DNA damage response posttranscriptional RNA operon in lymphocytes

Blood ◽

10.1182/blood-2010-09-310987 ◽

2011 ◽

Vol 117 (8) ◽

pp. 2441-2450 ◽

Cited By ~ 27

Author(s):

Krystyna Mazan-Mamczarz ◽

Patrick R. Hagner ◽

Yongqing Zhang ◽

Bojie Dai ◽

Elin Lehrmann ◽

...

Keyword(s):

Gene Expression ◽

Dna Damage ◽

Dna Damage Response ◽

Ataxia Telangiectasia ◽

Rna Binding ◽

Critical Role ◽

Cell Cycle Checkpoints ◽

Dependent Manner ◽

Damage Response ◽

Multiple Cell

Abstract Maintenance of genomic stability depends on the DNA damage response, a biologic barrier in early stages of cancer development. Failure of this response results in genomic instability and high predisposition toward lymphoma, as seen in patients with ataxia-telangiectasia mutated (ATM) dysfunction. ATM activates multiple cell-cycle checkpoints and DNA repair after DNA damage, but its influence on posttranscriptional gene expression has not been examined on a global level. We show that ionizing radiation modulates the dynamic association of the RNA-binding protein HuR with target mRNAs in an ATM-dependent manner, potentially coordinating the genotoxic response as an RNA operon. Pharmacologic ATM inhibition and use of ATM-null cells revealed a critical role for ATM in this process. Numerous mRNAs encoding cancer-related proteins were differentially associated with HuR depending on the functional state of ATM, in turn affecting expression of encoded proteins. The findings presented here reveal a previously unidentified role of ATM in controlling gene expression posttranscriptionally. Dysregulation of this DNA damage response RNA operon is probably relevant to lymphoma development in ataxia-telangiectasia persons. These novel RNA regulatory modules and genetic networks provide critical insight into the function of ATM in oncogenesis.

Download Full-text