A systems study reveals concurrent activation of AMPK and mTOR by amino acids.

Amino acids (aa) are not only building blocks for proteins, but also signalling molecules, with the mammalian target of rapamycin complex 1 (mTORC1) acting as a key mediator. However, little is known about whether aa, independently of mTORC1, activate other kinases of the mTOR signalling network. To delineate aa-stimulated mTOR network dynamics, we here combine a computational-experimental approach with text mining-enhanced quantitative proteomics. We report that AMP-activated protein kinase (AMPK), phosphatidylinositide 3-kinase (PI3K) and mTOR complex 2 (mTORC2) are acutely activated by aa-readdition in an mTORC1-independent manner. AMPK activation by aa is mediated by Ca2+/calmodulin-dependent protein kinase kinase ß (CaMKKß). In response, AMPK impinges on the autophagy regulators Unc-51-like kinase-1 (ULK1) and c-Jun. AMPK is widely recognized as an mTORC1 antagonist that is activated by starvation. We find that aa acutely activate AMPK concurrently with mTOR. We show that AMPK under aa sufficiency acts to sustain autophagy. This may be required to maintain protein homoeostasis and deliver metabolite intermediates for biosynthetic processes.

TSC1 activates TGF-ß-Smad2/3 signaling in growth arrest and epithelial-to-mesenchymal transition.

The tuberous sclerosis proteins TSC1 and TSC2 are key integrators of growth factor signaling. They suppress cell growth and proliferation by acting in a heteromeric complex to inhibit the mammalian target of rapamycin complex 1 (mTORC1). In this study, we identify TSC1 as a component of the transforming growth factor ß (TGF-ß)-Smad2/3 pathway. Here, TSC1 functions independently of TSC2. TSC1 interacts with the TGF-ß receptor complex and Smad2/3 and is required for their association with one another. TSC1 regulates TGF-ß-induced Smad2/3 phosphorylation and target gene expression and controls TGF-ß-induced growth arrest and epithelial-to-mesenchymal transition (EMT). Hyperactive Akt specifically activates TSC1-dependent cytostatic Smad signaling to induce growth arrest. Thus, TSC1 couples Akt activity to TGF-ß-Smad2/3 signaling. This has implications for cancer treatments targeting phosphoinositide 3-kinases and Akt because they may impair tumor-suppressive cytostatic TGF-ß signaling by inhibiting Akt- and TSC1-dependent Smad activation.

A dynamic network model of mTOR signaling reveals TSC-independent mTORC2 regulation.

The kinase mammalian target of rapamycin (mTOR) exists in two multiprotein complexes (mTORC1 and mTORC2) and is a central regulator of growth and metabolism. Insulin activation of mTORC1, mediated by phosphoinositide 3-kinase (PI3K), Akt, and the inhibitory tuberous sclerosis complex 1/2 (TSC1-TSC2), initiates a negative feedback loop that ultimately inhibits PI3K. We present a data-driven dynamic insulin-mTOR network model that integrates the entire core network and used this model to investigate the less well understood mechanisms by which insulin regulates mTORC2. By analyzing the effects of perturbations targeting several levels within the network in silico and experimentally, we found that, in contrast to current hypotheses, the TSC1-TSC2 complex was not a direct or indirect (acting through the negative feedback loop) regulator of mTORC2. Although mTORC2 activation required active PI3K, this was not affected by the negative feedback loop. Therefore, we propose an mTORC2 activation pathway through a PI3K variant that is insensitive to the negative feedback loop that regulates mTORC1. This putative pathway predicts that mTORC2 would be refractory to Akt, which inhibits TSC1-TSC2, and, indeed, we found that mTORC2 was insensitive to constitutive Akt activation in several cell types. Our results suggest that a previously unknown network structure connects mTORC2 to its upstream cues and clarifies which molecular connectors contribute to mTORC2 activation.

A modelling-experimental approach reveals insulin receptor substrate (IRS)-dependent regulation of adenosine monosphosphate-dependent kinase (AMPK) by insulin.

Mammalian target of rapamycin (mTOR) kinase responds to growth factors, nutrients and cellular energy status and is a central controller of cellular growth. mTOR exists in two multiprotein complexes that are embedded into a complex signalling network. Adenosine monophosphate-dependent kinase (AMPK) is activated by energy deprivation and shuts off adenosine 5'-triphosphate (ATP)-consuming anabolic processes, in part via the inactivation of mTORC1. Surprisingly, we observed that AMPK not only responds to energy deprivation but can also be activated by insulin, and is further induced in mTORC1-deficient cells. We have recently modelled the mTOR network, covering both mTOR complexes and their insulin and nutrient inputs. In the present study we extended the network by an AMPK module to generate the to date most comprehensive data-driven dynamic AMPK-mTOR network model. In order to define the intersection via which AMPK is activated by the insulin network, we compared simulations for six different hypothetical model structures to our observed AMPK dynamics. Hypotheses ranking suggested that the most probable intersection between insulin and AMPK was the insulin receptor substrate (IRS) and that the effects of canonical IRS downstream cues on AMPK would be mediated via an mTORC1-driven negative-feedback loop. We tested these predictions experimentally in multiple set-ups, where we inhibited or induced players along the insulin-mTORC1 signalling axis and observed AMPK induction or inhibition. We confirmed the identified model and therefore report a novel connection within the insulin-mTOR-AMPK network: we conclude that AMPK is positively regulated by IRS and can be inhibited via the negative-feedback loop.