Import of extracellular ATP in yeast and man modulates AMPK and TORC1 signalling.

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.

Palladium-Mediated Synthesis of a Near-Infrared Fluorescent K+ Sensor.

Potassium (K+) exits electrically excitable cells during normal and pathophysiological activity. Currently, K+-sensitive electrodes and electrical measurements are the primary tools to detect K+ fluxes. Here, we describe the synthesis of a near-IR, oxazine fluorescent K+ sensor (KNIR-1) with a dissociation constant suited for detecting changes in intracellular and extracellular K+ concentrations. KNIR-1 treatment of cells expressing voltage-gated K+ channels enabled the visualization of intracellular K+ depletion upon channel opening and restoration of cytoplasmic K+ after channel closing.

4,4'-Bismoschamine: biomimetic synthesis and evidence to support structural equivalency to montamine.

The dimeric natural product montamine was originally reported as two N-feruloylserotonin (moschamine) units linked by a nitrogen-nitrogen bond, but our recent synthesis of this symmetrical diacyl hydrazide structure revealed this to be incorrect. We subsequently hypothesized that the moschamine subunits were linked through the indole C4 site and that montamine was structurally identical to 4,4'-bismoschamine, a known natural product present in safflower oil. However, given that authentic samples of both montamine and 4,4'-bismoschamine were unavailable and that the NMR data for the natural products were recorded in different solvents, we were unable to unequivocally prove this hypothesis. A recent publication that claims montamine and 4,4'-bismoschamine are not the same natural product prompts us to disclose our own findings on this matter. A biomimetic synthesis of 4,4'-bismoschamine was developed that hinged on oxidative coupling of N-Boc-serotonin followed by elaboration of the resulting 4,4'-dimer to the natural product. A detailed comparison of the NMR data for synthetic 4,4'-bismoschamine with that reported for montamine revealed that while the 1H NMR data were in good agreement, the 13C NMR data displayed some discrepancies. In light of this result, the NMR data for several literature compounds was analyzed, the results of which revealed that the upfield chemical shifts of the methylene protons in the 1H NMR of montamine is unique to 4,4'-bistryptamines, supporting our initial statement that montamine and 4,4'-bismoschamine are structurally equivalent. Given that the main differences in the 13C NMR data between montamine and synthetic 4,4'-bismoschamine occur at the quaternary carbons, we propose that these peaks have been misassigned from a 13C NMR spectrum that was obtained from an impure sample and/or the small amount of montamine (4 mg) isolated from the natural source.

Total synthesis of putative montamine and a proposed structural reassignment.

The natural product montamine was originally assigned as a homodimer of moschamine linked by a N-N' bond at the serotonin side-chain. A total synthesis of the reported structure has shown this to be incorrect. Analysis of the spectroscopic data suggests that the dimerization site has been incorrectly assigned, and montamine is likely to be a 4,4'-bismoschamine natural product previously described in the literature.

mTORC1 directly inhibits AMPK to promote cell proliferation under nutrient stress.

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.

Nitrogen regulates AMPK to control TORC1 signaling.

BACKGROUND: Cell 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. RESULTS: Here, 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 ß 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 AMPKα(Ssp2) T loop. However, we find that a second homolog CaMKK(Ppk34) is specifically required to stimulate AMPKα(Ssp2) 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. CONCLUSIONS: The alternative nitrogen source ammonia can simulate TORC1 activity to support growth and division under challenging nutrient settings, a situation often seen in cancer.

Environmental control of cell size at division.

Tight coupling between cell growth and cell cycle progression allows cells to adjust their size to the demands of proliferation in varying nutrient environments. Target of rapamycin (TOR) signalling pathways co-ordinate cell growth with cell cycle progression in response to altered nutritional availability. To increase cell size the active TOR Complex 1 (TORC1) promotes cell growth to delay mitosis and cell division, whereas under limited nutrients TORC1 activity is decreased to reduce cell size. It remains unclear why cell size is subject to such tight control. Recent evidence suggests that in addition to modulating cell size, changes in nutrient availability also alter nuclear:cytoplasmic (N/C) ratios and may therefore compromise optimal cellular physiology. This could explain why cells increase their size when conditions are favourable, despite being competent to survive at a smaller size if necessary.