Abstract
cAMP and mTOR signalling pathways control a number of critical cellular processes including metabolism, protein synthesis, proliferation and cell survival and therefore understanding the signalling events which integrate these two signalling pathways is of particular interest. In this study, we show that the pharmacological elevation of [cAMP]i in mouse embryonic fibroblasts (MEFs) and human embryonic kidney 293 (HEK293) cells inhibits mTORC1 activation via a PKA-dependent mechanism. Although the inhibitory effect of cAMP on mTOR could be mediated by impinging on signalling cascades (i.e. PKB, MAPK and AMPK) that inhibit TSC1/2, an upstream negative regulator of mTORC1, we show that cAMP inhibits mTORC1 in TSC2 knockout (TSC2−/−) MEFs. We also show that cAMP inhibits insulin and amino acid-stimulated mTORC1 activation independently of Rheb, Rag GTPases, TSC2, PKB, MAPK and AMPK, indicating that cAMP may act independently of known regulatory inputs into mTOR. Moreover, we show that the prolonged elevation in [cAMP]i can also inhibit mTORC2. We provide evidence that this cAMP-dependent inhibition of mTORC1/2 is caused by the dissociation of mTORC1 and 2 and a reduction in mTOR catalytic activity, as determined by its auto-phosphorylation on Ser2481. Taken together, these results provide an important insight into how cAMP signals to mTOR and down-regulates its activity, which may lead to the identification of novel drug targets to inhibit mTOR that could be used for the treatment and prevention of human diseases such as cancer.
Highlights
Mammalian target of rapamycin is a Ser/Thr protein kinase that exists in two biochemically and functionally distinct multi-component complexes known as mTORC1 and mTORC2 [1,2]. mTORC1 couples nutrient availability with hormonal and growth factor signals to regulate metabolism, cell growth and proliferation [3,4], and phosphorylates a number of proteins involved in protein translation including ribosomal protein S6 kinases 1 and 2 (S6K1/2) and eukaryotic initiation factor 4E binding proteins (4EBPs) [5]. mTORC1 activity is regulated by numerous signalling pathways, many of which converge on tuberous sclerosis complex 1 and 2 (TSC1/2) [3]
The second messenger cAMP is yielded in response to a broad range of extracellular stimuli that act upon G-protein coupled receptors (GPCR) [9]. cAMP is synthesized by the action of adenylate cyclase (AC) and its degradation is mediated by the action of cAMP phosphodiesterases (PDEs) [10,11]
In this report we demonstrate, in mouse embryonic fibroblasts (MEFs) and human embryonic kidney cells (HEK293), that cAMP signals to and inhibits mTORC1/2 via their dissociation and a decrease in mTOR intrinsic catalytic activity
Summary
Mammalian target of rapamycin (mTOR, known as FRAP, RAFT or RAPT) is a Ser/Thr protein kinase that exists in two biochemically and functionally distinct multi-component complexes known as mTORC1 and mTORC2 [1,2]. mTORC1 couples nutrient availability with hormonal and growth factor signals to regulate metabolism, cell growth and proliferation [3,4], and phosphorylates a number of proteins involved in protein translation including ribosomal protein S6 kinases 1 and 2 (S6K1/2) and eukaryotic initiation factor 4E binding proteins (4EBPs) [5]. mTORC1 activity is regulated by numerous signalling pathways, many of which converge on tuberous sclerosis complex 1 and 2 (TSC1/2) [3]. CAMP can either stimulate [15,16,17,18,19,20] or inhibit [21,22,23,24,25,26] mTORC1 depending upon cell type This is likely through the activation or suppression of signalling transduction cascades upstream of mTORC1 such as the PKB [16,26,27,28,29], the MAPK (mitogenactivated protein kinase) [30] and the AMPK Glucagon, a GsPCR agonist that increase [cAMP]i, inhibits mTORC1 in rat hepatocytes [23,25] This is coincident with an increase in AMPK phosphorylation on Thr172 [23,25], a positive regulator of TSC1/2 [33]. This report provides new insights into how cAMP talks to mTOR and could lead to the discovery of new anti-cancer drug targets
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