CDK7 is a component of the integrated stress response regulating SNAT2 (SLC38A2)/System A adaptation in response to cellular amino acid deprivation

Clare Stretton, Christopher Lipina, Russell Hyde, Emma Cwiklinski, Thorsten M. Hoffmann, Peter M. Taylor, Harinder S. Hundal (Lead / Corresponding author)

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Abstract

Extracellular amino acid (AA) withdrawal/restriction invokes an integrated stress response (ISR) that induces global suppression of protein synthesis whilst allowing transcription and translation of a select group of genes, whose protein products facilitate cellular adaptation to AA insufficiency. Transcriptional induction of the System A/SNAT2 AA transporter represents a classic adaptation response and crucially depends upon activation of the General Control Nonderepressible-2 kinase/Activating transcription factor 4 (GCN2/ATF4) pathway. However, the ISR may also include additional signalling inputs operating in conjunction or independently of GCN2/ATF4 to upregulate SNAT2. Herein, we show that whilst pharmacological inhibition of MEK-ERK, mTORC1 and p38 MAP kinase signalling has no detectable effect on System A upregulation, inhibitors targeting GSK3 (e.g. SB415286) caused significant repression of the SNAT2 adaptation response. Strikingly, the effects of SB415286 persist in cells in which GSK3α/β have been stably silenced indicating an off-target effect. We show that SB415286 can also inhibit cyclin-dependent kinases (CDK) and that roscovitine and flavopiridol (two pan CDK inhibitors) are effective repressors of the SNAT2 adaptive response. In particular, our work reveals that CDK7 activity is upregulated in AA-deprived cells in a GCN-2-dependent manner and that a potent and selective CDK7 inhibitor, THZ-1, not only attenuates the increase in ATF4 expression but blocks System A adaptation. Importantly, the inhibitory effects of THZ-1 on System A adaptation are mitigated in cells expressing a doxycycline-inducible drug-resistant form of CDK7. Our data identify CDK7 as a novel component of the ISR regulating System A adaptation in response to AA insufficiency.

Original languageEnglish
Pages (from-to)978-991
Number of pages14
JournalBiochimica et Biophysica Acta. Molecular Cell Research
Volume1866
Issue number6
Early online date8 Mar 2019
DOIs
Publication statusPublished - 1 Jun 2019

Fingerprint

Activating Transcription Factor 4
Amino Acids
Cyclin-Dependent Kinases
alvocidib
Phosphotransferases
Up-Regulation
Amino Acid Transport Systems
Doxycycline
Mitogen-Activated Protein Kinase Kinases
Extracellular Signal-Regulated MAP Kinases
p38 Mitogen-Activated Protein Kinases
Proteins
Pharmacology
Pharmaceutical Preparations
3-(3-chloro-4-hydroxyphenylamino)-4-(4-nitrophenyl)-1H-pyrrole-2,5-dione

Keywords

  • ATF4
  • GCN2
  • GSK3
  • Me-AIB
  • Roscovitine
  • Transporter

Cite this

@article{a63e73e27b984fd891e5289575bec6dc,
title = "CDK7 is a component of the integrated stress response regulating SNAT2 (SLC38A2)/System A adaptation in response to cellular amino acid deprivation",
abstract = "Extracellular amino acid (AA) withdrawal/restriction invokes an integrated stress response (ISR) that induces global suppression of protein synthesis whilst allowing transcription and translation of a select group of genes, whose protein products facilitate cellular adaptation to AA insufficiency. Transcriptional induction of the System A/SNAT2 AA transporter represents a classic adaptation response and crucially depends upon activation of the General Control Nonderepressible-2 kinase/Activating transcription factor 4 (GCN2/ATF4) pathway. However, the ISR may also include additional signalling inputs operating in conjunction or independently of GCN2/ATF4 to upregulate SNAT2. Herein, we show that whilst pharmacological inhibition of MEK-ERK, mTORC1 and p38 MAP kinase signalling has no detectable effect on System A upregulation, inhibitors targeting GSK3 (e.g. SB415286) caused significant repression of the SNAT2 adaptation response. Strikingly, the effects of SB415286 persist in cells in which GSK3α/β have been stably silenced indicating an off-target effect. We show that SB415286 can also inhibit cyclin-dependent kinases (CDK) and that roscovitine and flavopiridol (two pan CDK inhibitors) are effective repressors of the SNAT2 adaptive response. In particular, our work reveals that CDK7 activity is upregulated in AA-deprived cells in a GCN-2-dependent manner and that a potent and selective CDK7 inhibitor, THZ-1, not only attenuates the increase in ATF4 expression but blocks System A adaptation. Importantly, the inhibitory effects of THZ-1 on System A adaptation are mitigated in cells expressing a doxycycline-inducible drug-resistant form of CDK7. Our data identify CDK7 as a novel component of the ISR regulating System A adaptation in response to AA insufficiency.",
keywords = "ATF4, GCN2, GSK3, Me-AIB, Roscovitine, Transporter",
author = "Clare Stretton and Christopher Lipina and Russell Hyde and Emma Cwiklinski and Hoffmann, {Thorsten M.} and Taylor, {Peter M.} and Hundal, {Harinder S.}",
note = "This work was supported by the Biotechnology and Biological Sciences Research Council and Diabetes UK. We are extremely grateful to Dr. Nathaneal Gray (Harvard Medical School) for providing HeLa S3 cells stably carrying the doxycycline-inducible drug-resistant Flag-CDK7 C312S construct and for the generous gift of THZ-531.",
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CDK7 is a component of the integrated stress response regulating SNAT2 (SLC38A2)/System A adaptation in response to cellular amino acid deprivation. / Stretton, Clare; Lipina, Christopher; Hyde, Russell; Cwiklinski, Emma; Hoffmann, Thorsten M.; Taylor, Peter M.; Hundal, Harinder S. (Lead / Corresponding author).

In: Biochimica et Biophysica Acta. Molecular Cell Research, Vol. 1866, No. 6, 01.06.2019, p. 978-991.

Research output: Contribution to journalArticle

TY - JOUR

T1 - CDK7 is a component of the integrated stress response regulating SNAT2 (SLC38A2)/System A adaptation in response to cellular amino acid deprivation

AU - Stretton, Clare

AU - Lipina, Christopher

AU - Hyde, Russell

AU - Cwiklinski, Emma

AU - Hoffmann, Thorsten M.

AU - Taylor, Peter M.

AU - Hundal, Harinder S.

N1 - This work was supported by the Biotechnology and Biological Sciences Research Council and Diabetes UK. We are extremely grateful to Dr. Nathaneal Gray (Harvard Medical School) for providing HeLa S3 cells stably carrying the doxycycline-inducible drug-resistant Flag-CDK7 C312S construct and for the generous gift of THZ-531.

PY - 2019/6/1

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N2 - Extracellular amino acid (AA) withdrawal/restriction invokes an integrated stress response (ISR) that induces global suppression of protein synthesis whilst allowing transcription and translation of a select group of genes, whose protein products facilitate cellular adaptation to AA insufficiency. Transcriptional induction of the System A/SNAT2 AA transporter represents a classic adaptation response and crucially depends upon activation of the General Control Nonderepressible-2 kinase/Activating transcription factor 4 (GCN2/ATF4) pathway. However, the ISR may also include additional signalling inputs operating in conjunction or independently of GCN2/ATF4 to upregulate SNAT2. Herein, we show that whilst pharmacological inhibition of MEK-ERK, mTORC1 and p38 MAP kinase signalling has no detectable effect on System A upregulation, inhibitors targeting GSK3 (e.g. SB415286) caused significant repression of the SNAT2 adaptation response. Strikingly, the effects of SB415286 persist in cells in which GSK3α/β have been stably silenced indicating an off-target effect. We show that SB415286 can also inhibit cyclin-dependent kinases (CDK) and that roscovitine and flavopiridol (two pan CDK inhibitors) are effective repressors of the SNAT2 adaptive response. In particular, our work reveals that CDK7 activity is upregulated in AA-deprived cells in a GCN-2-dependent manner and that a potent and selective CDK7 inhibitor, THZ-1, not only attenuates the increase in ATF4 expression but blocks System A adaptation. Importantly, the inhibitory effects of THZ-1 on System A adaptation are mitigated in cells expressing a doxycycline-inducible drug-resistant form of CDK7. Our data identify CDK7 as a novel component of the ISR regulating System A adaptation in response to AA insufficiency.

AB - Extracellular amino acid (AA) withdrawal/restriction invokes an integrated stress response (ISR) that induces global suppression of protein synthesis whilst allowing transcription and translation of a select group of genes, whose protein products facilitate cellular adaptation to AA insufficiency. Transcriptional induction of the System A/SNAT2 AA transporter represents a classic adaptation response and crucially depends upon activation of the General Control Nonderepressible-2 kinase/Activating transcription factor 4 (GCN2/ATF4) pathway. However, the ISR may also include additional signalling inputs operating in conjunction or independently of GCN2/ATF4 to upregulate SNAT2. Herein, we show that whilst pharmacological inhibition of MEK-ERK, mTORC1 and p38 MAP kinase signalling has no detectable effect on System A upregulation, inhibitors targeting GSK3 (e.g. SB415286) caused significant repression of the SNAT2 adaptation response. Strikingly, the effects of SB415286 persist in cells in which GSK3α/β have been stably silenced indicating an off-target effect. We show that SB415286 can also inhibit cyclin-dependent kinases (CDK) and that roscovitine and flavopiridol (two pan CDK inhibitors) are effective repressors of the SNAT2 adaptive response. In particular, our work reveals that CDK7 activity is upregulated in AA-deprived cells in a GCN-2-dependent manner and that a potent and selective CDK7 inhibitor, THZ-1, not only attenuates the increase in ATF4 expression but blocks System A adaptation. Importantly, the inhibitory effects of THZ-1 on System A adaptation are mitigated in cells expressing a doxycycline-inducible drug-resistant form of CDK7. Our data identify CDK7 as a novel component of the ISR regulating System A adaptation in response to AA insufficiency.

KW - ATF4

KW - GCN2

KW - GSK3

KW - Me-AIB

KW - Roscovitine

KW - Transporter

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VL - 1866

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