• Mon. May 23rd, 2022

Whether GCN2 physically interacts with CDK7 or can influence phosphorylation of the latter on either one of its two T-loop residues (Ser164 or Thr170) that are crucial for the kinase to form a stable complex with cyclin H and MAT1 and for its CTD kinase activity [30] remain interesting, but open questions for future investigation

Byacusticavisual

Jan 28, 2022

Whether GCN2 physically interacts with CDK7 or can influence phosphorylation of the latter on either one of its two T-loop residues (Ser164 or Thr170) that are crucial for the kinase to form a stable complex with cyclin H and MAT1 and for its CTD kinase activity [30] remain interesting, but open questions for future investigation. Conflict of interests None of the authors have any conflict of interests to declare in relation to the current study. Author contributions CS, CL, RH, EC and TH were involved in performing the experiments and data curation; CS, PT, HSH Fosdagrocorat were involved in conceptualization and formal analysis. pharmacological inhibition of MEK-ERK, mTORC1 and p38 MAP kinase signalling has no detectable effect on System A upregulation, inhibitors targeting GSK3 (SB415286) caused significant repression of the SNAT2 adaptation response. Strikingly, the effects of SB415286 persist in cells in which GSK3/ 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 Fosdagrocorat 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. SLC38A1, SLC38A2 and SLC38A4, respectively) and these mediate the sodium-dependent uptake of short chain neutral AAs such as alanine, serine and threonine. System A was functionally characterised by its ability to accept N-alkylated substrates such as -(methyl-amino)isobutyric acid (MeAIB), whereas, those of the System N family, which include SNAT3, SNAT5 and SNAT7 (SLC38A3, SLC38A5 and SLC38A7 respectively), do not accept Me-AIB but show preference for AAs Fosdagrocorat containing an extra nitrogen in their side chains (glutamine, asparagine and histidine) as substrates and, moreover, exhibit tolerance for lithium as a sodium substitute [26]. Whilst transporters of the System A sub group share significant sequence homology, it is widely established that SNAT2 (SLC38A2) is the most ubiquitously expressed and, strikingly, one of the most extensively regulated AA transporters to have been documented to date, possibly reflecting its important contribution to cellular AA nutrition and to the control of diverse cellular functions. SNAT2 expression/activity is, for example, subject to both acute and chronic modulation by hormones (glucocorticoids, estrogen, insulin) and growth factors [2,20,24,55]. In tissues, such as the mammary gland, the transcriptional upregulation of SNAT2 by 17-estradiol may play a significant role in meeting the increased AA demand that facilitates differentiation and proliferation of this tissue in preparation for lactation [55], whereas, in skeletal muscle, recruitment of SNAT2 carriers from an intracellular compartment to the plasma membrane and the attendant increase in AA delivery in response to insulin may form part of the anabolic effect that the hormone has upon muscle protein synthesis [20,24]. SNAT2 can also be upregulated in cells subjected to hyperosmotic stress; a response designed to elevate cellular intake of organic osmolytes (AAs) that helps establish an osmotic drive for water uptake into cells to restore both intracellular volume and ionic strength [6,10,36]. Crucially, the sodium coupled uptake of extracellular AAs establishes an outwardly-directed concentration gradient of SNAT substrates, which, if not immediately utilised for metabolic processes, can leave the cell tertiary Fosdagrocorat exchange transporters, such as the leucine-preferring (LAT1) carrier, that operates in parallel with SNAT2 in the plasma membrane MMP15 [5,21]. This SNAT2/LAT1 exchange coupling is considered significant for intracellular leucine delivery given that this essential AA serves to potently activate the mTORC1/S6K1 signalling axis [33]. The mechanistic target of rapamycin complex 1 (mTORC1) plays a pivotal role in the control of mRNA translation, cell growth/metabolism and autophagy [50] and consequently factors affecting SNAT2 expression/activity will indirectly impact on the regulation of these key cellular processes by virtue of the changes that occur in mTORC1 activity [47,54]. Whilst AA insufficiency, even of a single AA such as methionine or leucine, exerts a profound suppressive effect on global mRNA translation [37], the expression and translation of a sub-set of genes that allow cellular adaptation to changes in environmental nutrient supply is upregulated.