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SummaryBackgroundCell growth and cell-cycle progression are tightly coordinated to enable cells to adjust their size (timing of division) to the demands of proliferation in varying nutritional environments. In fission yeast, nitrogen stress results in sustained proliferation at a reduced size.ResultsHere, we show that cells can sense nitrogen stress to reduce target of rapamycin complex-1 (TORC1) activity. Nitrogen-stress-induced TORC1 inhibition differs from amino-acid-dependent control of TORC1 and requires the Ssp2 (AMPKα) kinase, the Tsc1/2 complex, and Rhb1 GTPase. Importantly, the β and γ regulatory subunits of AMPK are not required to control cell division in response to nitrogen stress, providing evidence for a nitrogen-sensing mechanism that is independent of changes in intracellular ATP/AMP levels. The CaMKK homolog Ssp1 is constitutively required for phosphorylation of the AMPKαSsp2 T loop. However, we find that a second homolog CaMKKPpk34 is specifically required to stimulate AMPKαSsp2 activation in response to nitrogen stress. Finally, ammonia also controls mTORC1 activity in human cells; mTORC1 is activated upon the addition of ammonium to glutamine-starved Hep3B cancer cells.ConclusionsThe alternative nitrogen source ammonia can simulate TORC1 activity to support growth and division under challenging nutrient settings, a situation often seen in cancer.

Central to cellular metabolism and cell proliferation are highly conserved signalling pathways controlled by mammalian target of rapamycin (mTOR) and AMP-activated protein kinase (AMPK) 1 , 2 , dysregulation of which are implicated in pathogenesis of major human diseases such as cancer and type 2 diabetes. AMPK pathways leading to reduced cell proliferation are well established and, in part, act through inhibition of TOR complex-1 (TORC1) activity. Here we demonstrate reciprocal regulation, specifically that TORC1 directly down-regulates AMPK signalling by phosphorylating the evolutionarily conserved residue Ser367 in the fission yeast AMPK catalytic subunit Ssp2, and AMPK α1Ser347/α2Ser345 in the mammalian homologs, which is associated with reduced phosphorylation of activation loop Thr172. Genetic or pharmacological inhibition of TORC1 signalling led to AMPK activation in the absence of increased AMP:ATP ratios; under nutrient stress conditions this was associated with growth limitation in both yeast and human cell cultures. Our findings reveal fundamental, bi-directional regulation between two major metabolic signalling networks and uncover new opportunity for cancer treatment strategies aimed at suppressing cell proliferation in the nutrient-poor tumor microenvironment.

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AMP-activated kinase (AMPK) and target of rapamycin (TOR) signalling coordinate cell growth, proliferation, metabolism and cell survival with the nutrient environment of cells. The poor vasculature and nutritional stress experienced by cells in solid tumours raises the question: how do they assimilate sufficient nutrients to survive? Here, we show that human and fission yeast cells import ATP and AMP from their external environment to regulate AMPK and TOR signalling. Exposure of fission yeast ( Schizosaccharomyces pombe ) and human cells to external AMP impeded cell growth; however, in yeast this restraining impact required AMPK. In contrast, external ATP rescued the growth defect of yeast mutants with reduced TORC1 signalling; furthermore, exogenous ATP transiently enhanced TORC1 signalling in both yeast and human cell lines. Addition of the PANX1 channel inhibitor probenecid blocked ATP import into human cell lines suggesting that this channel may be responsible for both ATP release and uptake in mammals. In light of these findings, it is possible that the higher extracellular ATP concentration reported in solid tumours is both scavenged and recognized as an additional energy source beneficial for cell growth.