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.
Highlights
The solute carrier 38 (SLC38) transporter family comprises six functionally characterised amino acid (AA) transporters from the eleven members that have been identified in the human genome and notably include two sub groups whose activities align with those classically described as System A or System N [9]
In the current investigation we show that whilst inhibition of MEKERK, mechanistic target of rapamycin complex 1 (mTORC1) and p38 MAP kinase signalling have no impact on the adaptive increase in SNAT2 activity induced by AA insufficiency, two structurally unrelated Glycogen Synthase Kinase-3 (GSK3) inhibitors significantly blunt the SNAT2 response to AA withdrawal
Anti-Slc38a2 (SNAT2) antibody was from MBL, anti-α-subunit Na/K-ATPase antibody was from the Developmental Studies Hybridoma Bank (University of Iowa), anti-β actin, anti-GAPDH and anti-Flag antibodies were from Sigma-Aldrich, anti-V5 and Alexa-Fluor488 anti-mouse antibodies were from Invitrogen
Summary
The solute carrier 38 (SLC38) transporter family comprises six functionally characterised amino acid (AA) transporters from the eleven members that have been identified in the human genome and notably include two sub groups whose activities align with those classically described as System A or System N [9]. System A was functionally characterised by its ability to accept N-alkylated substrates such as α-(methyl-amino)isobutyric acid (MeAIB), whereas, those of the System N family, which include SNAT3, SNAT5 and SNAT7 (aka SLC38A3, SLC38A5 and SLC38A7 respectively), do not accept Me-AIB but show preference for AAs containing an extra nitrogen in their side chains (i.e. glutamine, asparagine and histidine) as substrates and, exhibit tolerance for lithium as a sodium substitute [26]. SNAT2 expression/activity is, for example, subject to both acute and chronic modulation by hormones (e.g. glucocorticoids, estrogen, insulin) and growth factors [2,20,24,55]. In tissues, such as the mammary gland, the transcriptional upregulation of SNAT2 by 17β-estradiol may play a significant
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